Methods of reducing polysorbate degradation in drug formulations

ABSTRACT

The present disclosure pertains to compositions with reduced residual amount of lipases and methods of making such compositions. In particular, it pertains to compositions and methods of such making compositions by depleting the compositions of certain lipases, such as, liver carboxylesterase B-1-like protein and liver carboxylesterase 1-like protein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationNo. 62/982,346, filed Feb. 27, 2020, U.S. Provisional Patent ApplicationNo. 63/021,181, filed May 7, 2020 and U.S. Provisional PatentApplication No. 63/073,125, filed Sep. 1, 2020, the contents of whichare incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on May 18, 2021, isnamed 070816-01942_SL.txt and is 20,608 bytes in size.

FIELD

The present invention generally pertains to compositions with reducedamount of certain lipases, methods of making such compositions andmethods of reducing polysorbate degradation due to the presence of suchlipases. In particular, the present invention generally pertains tocompositions and methods of making compositions with reduced presence ofliver carboxylesterase-B 1-like protein and livercarboxylesterase-1-like protein.

BACKGROUND

Among drug products, protein-based biotherapeutics are an importantclass of drugs that offer a high level of selectivity, potency andefficacy, as evidenced by the considerable increase in clinical trialswith monoclonal antibodies (mAbs) over the past several years. Bringinga protein-based biotherapeutic to the clinic can be a multiyearundertaking requiring coordinated efforts throughout various researchand development disciplines, including discovery, process andformulation development, analytical characterization, and pre-clinicaltoxicology and pharmacology.

One critical aspect for a clinically and commercially viablebiotherapeutic is stability of the drug product in terms of themanufacturing process as well as shelf life. This often necessitatesappropriate steps to help increase physical and chemical stability ofthe protein-based biotherapeutics throughout the different solutionconditions and environments necessary for manufacturing and storage withminimal impact on product quality, including identifying molecules withgreater inherent stability, protein engineering, and formulationdevelopment. Surfactants, such as, polysorbate are often used to enhancethe physical stability of a protein-based biotherapeutic product. Overseventy percent of marketed monoclonal antibody therapeutics containbetween 0.001% and 0.1% polysorbate, a type of surfactant, to impartphysical stability to the protein-based biotherapeutics. Polysorbatesare susceptible to auto-oxidation and hydrolysis, which results in freefatty acids and subsequent fatty acid particle formation. Thedegradation of polysorbate can adversely affect the drug product qualitysince polysorbate can protect against interfacial stress, such asaggregation and adsorption. Presence of some lipases can be a likelycause of degradation of polysorbates in a formulation. Thus, suchlipases in drug products need to be detected, monitored and reduced.

Direct analysis of lipases can require isolation of the product in asufficiently large amount for the assay, which is undesirable and hasonly been possible in selected cases. Hence, it is a challenging task todetermine the workflow and analytical tests required to characterizelipases responsible for polysorbate degradation in a sample. In additionto detecting the lipases responsible for polysorbate degradation, thedrug product must be obtained by purification methods that remove orreduce such lipases.

It will be appreciated that a need exists for methods for depletinglipase from a formulated drug product.

SUMMARY

Maintaining stability of drug formulations, not only during storage butalso during manufacturing, shipment, handling and administration, is asignificant challenge. Among drug products, protein biotherapeutics aregaining popularity due to their success and versatility. One of themajor challenges for protein biotherapeutics development is to overcomethe limited stability of the protein and excipients in the products,which can be affected by the presence of lipases (present as host-cellproteins). Evaluation of its effect on the drug formulation andreduction of such lipases can be an important step in drug formulationdevelopment, followed by methods to prepare the drug formulation so asto have reduced lipases and increased stability owing to the reducedlipases.

In one exemplary embodiment, the disclosure provides a method ofdepleting lipase from a sample comprising contacting the sampleincluding lipase with a probe, said probe capable of binding to thelipase to form a complex and separating the complex from the sample tothereby deplete the lipase from the sample. In one aspect, the samplecan comprise a protein of interest. In one aspect, the sample cancomprise a polysorbate excipient. In a specific aspect, the polysorbateexcipient can be selected from polysorbate-20, polysorbate-60,polysorbate-80 or combinations thereof. In yet another specific aspect,the polysorbate excipient is polysorbate-80.

In one aspect, the lipase is liver carboxylesterase-B1-like protein. Inanother aspect, the lipase is liver carboxylesterase-1-like protein.

In one aspect, the lipase is capable of degrading the polysorbate in thesample. Thus, the method of this embodiment reduces the degradation ofpolysorbates by depleting the sample of the lipase.

In one aspect, the probe can be capable of being linked to a solidsupport. In a specific aspect, the solid support can be agarose beads ormagnetic beads.

In one aspect, the probe can be attached to a solid support using aligand. In a specific aspect, the ligand can be an indicator, biotinmolecule, a modified biotin molecule, a nuclei, a sequence, an epitopetag, an electron poor molecule or an electron rich molecule.

In one aspect, the method can further comprise recovering the lipasefrom the complex.

In one exemplary embodiment, the disclosure provides a method ofpurifying a sample having a protein of interest and a lipase, comprisingcontacting the sample with a probe, said probe capable of binding to thelipase to form a complex and separating the complex from the sample. Inone aspect, the lipase is liver carboxylesterase-B1-like protein. Inanother aspect, the lipase is liver carboxylesterase-1-like protein.

In one aspect, the sample comprises a polysorbate excipient. In aspecific aspect, the polysorbate excipient can be selected frompolysorbate-20, polysorbate-60, polysorbate-80 or combinations thereof.In yet another specific aspect, the polysorbate excipient can bepolysorbate-80.

In one aspect, the probe can be capable of being linked to a solidsupport. In a specific aspect, the solid support can be agarose beads ormagnetic beads.

In one aspect, the probe can be attached to a solid support using aligand. In a specific aspect, the ligand can be an indicator, biotinmolecule, a modified biotin molecule, a nuclei, a sequence, an epitopetag, an electron poor molecule or an electron rich molecule.

In one exemplary embodiment, the disclosure provides a method ofdecreasing degradation of polysorbate in a sample, comprising contactingthe sample including lipase and polysorbate with a probe, said probecapable of binding to the lipase to form a complex and separating thecomplex from the sample to thereby decrease degradation of polysorbatein the sample.

In one aspect, the lipase is liver carboxylesterase-B1-like protein. Inanother aspect, the lipase is liver carboxylesterase-1-like protein.

In one aspect, the sample can comprise a protein of interest. In oneaspect, the sample can comprise a polysorbate excipient. In a specificaspect, the polysorbate excipient is selected from polysorbate-20,polysorbate-60, polysorbate-80 or combinations thereof. In yet anotherspecific aspect, the polysorbate excipient is polysorbate-80.

In one aspect, the probe can be capable of being linked to a solidsupport. In a specific aspect, the solid support can be agarose beads ormagnetic beads.

In one aspect, the probe can be attached to a solid support using aligand. In a specific aspect, the ligand can be an indicator, biotinmolecule, a modified biotin molecule, a nuclei, a sequence, an epitopetag, an electron poor molecule or an electron rich molecule.

In one exemplary embodiment, the disclosure provides a compositioncomprising a protein of interest purified from mammalian cells and aresidual amount of liver carboxylesterase-B1-like protein. In oneaspect, the residual amount of liver carboxylesterase-B1-like protein isless than about 5 ppm. In another aspect, the composition can furthercomprise a surfactant. In yet a further aspect, the surfactant can be ahydrophilic nonionic surfactant. In another aspect, the surfactant canbe a sorbitan fatty acid ester. In a specific aspect, the surfactant canbe a polysorbate. In another specific aspect, the concentration of thepolysorbate in the composition can be about 0.01% w/v to about 0.2% w/v.In a further specific aspect, the surfactant can be a polysorbate 80. Inone aspect, the mammalian cells can include a CHO cell.

In one aspect, the liver carboxylesterase-B1-like protein can causedegradation of polysorbate 80.

In one aspect, the composition can be a parenteral formulation.

In one aspect, the protein of interest can be a monoclonal antibody, apolyclonal antibody, a bispecific antibody, an antibody fragment, afusion protein, or an antibody-drug complex. In one aspect, theconcentration of the protein of interest can be about 20 mg/mL to about400 mg/mL.

In one aspect, the composition can further comprise one or morepharmaceutically acceptable excipients. In another aspect, thecomposition can further comprise a buffer selected from a groupconsisting of histidine buffer, citrate buffer, alginate buffer, andarginine buffer. In one aspect, the composition can further comprise atonicity modifier. In yet another aspect, the composition can furthercomprise sodium phosphate.

In one exemplary embodiment, the disclosure provides a compositioncomprising a protein of interest purified from mammalian cells and aresidual amount of liver carboxylesterase-1-like protein. In one aspect,the residual amount of liver carboxylesterase-1-like protein is lessthan about 5 ppm. In another aspect, the composition can furthercomprise a surfactant. In yet a further aspect, the surfactant can be ahydrophilic nonionic surfactant. In another aspect, the surfactant canbe a sorbitan fatty acid ester. In a specific aspect, the surfactant canbe a polysorbate. In another specific aspect, the concentration of thepolysorbate in the composition can be about 0.01% w/v to about 0.2% w/v.In a further specific aspect, the surfactant can be a polysorbate 80. Inone aspect, the mammalian cells can include a CHO cell.

In one aspect, the liver carboxylesterase-1-like protein can causedegradation of polysorbate 80.

In one aspect, the composition can be a parenteral formulation.

In one aspect, the protein of interest can be a monoclonal antibody, apolyclonal antibody, a bispecific antibody, an antibody fragment, afusion protein, or an antibody-drug complex. In one aspect, theconcentration of the protein of interest can be about 20 mg/mL to about400 mg/mL.

In one aspect, the composition can further comprise one or morepharmaceutically acceptable excipients. In another aspect, thecomposition can further comprise a buffer selected from a groupconsisting of histidine buffer, citrate buffer, alginate buffer, andarginine buffer. In one aspect, the composition can further comprise atonicity modifier. In yet another aspect, the composition can furthercomprise sodium phosphate.

In one exemplary embodiment, the disclosure provides a method ofdetecting a lipase in a sample. In one aspect, the lipases can be livercarboxylesterase-1-like protein or liver carboxylesterase-B1-likeprotein. In one aspect, the method of detecting a lipase in a sample cancomprise contacting the sample with a serine hydrolase probe. In oneaspect, the method of detecting a lipase in a sample can comprisecontacting and incubating the sample with a serine hydrolase probe toform a complex of lipase and serine hydrolase probe. In a furtheraspect, the method of detecting a lipase in a sample can comprisefiltering out the serine hydrolase probe that does not form the complexof lipase and serine hydrolase probe.

In one aspect, the method of detecting a lipase in a sample can furthercomprise contacting the contacting the sample with magnetic beads havingan ability to bind to the serine hydrolase probe to such that magneticbeads are bound to the complex of lipase and serine hydrolase probe. Themagnetic beads bound to the complex of lipase and serine hydrolase probecan be further removed from the sample and washed with a buffer.

In another aspect, the method can further comprise removing the magneticbeads which are bound to the complex of lipase and serine hydrolaseprobe to form a solution of enriched lipases.

In one aspect, the method can further comprise adding hydrolyzing agentto the solution to obtain digests. In a specific aspect, the hydrolyzingagent can be trypsin. In one aspect, the method can further compriseanalyzing the digests to detect the lipases. In one aspect, the digestscan be analyzed using a mass spectrometer. In a specific aspect, themass spectrometer can be a tandem mass spectrometer. In another specificaspect, the mass spectrometer can be coupled to a liquid chromatographysystem. In yet another specific aspect, the mass spectrometer can becoupled to a liquid chromatography—multiple reaction monitoring system.

In one aspect, the method can further comprise adding protein denaturingagent to the solution. In a specific aspect, the protein denaturingagent can be urea. In one aspect, the method can further comprise addingprotein reducing agent to the solution. In a specific aspect, theprotein reducing agent can be DTT (dithiothreitol). In one aspect, themethod can further comprise adding protein alkylating agent to thesolution. In a specific aspect, the protein alkylating agent can beiodoacetamide.

These, and other, aspects of the invention will be better appreciatedand understood when considered in conjunction with the followingdescription and the accompanying drawings. The following description,while indicating various embodiments and numerous specific detailsthereof, is given by way of illustration and not of limitation. Manysubstitutions, modifications, additions, or rearrangements may be madewithin the scope of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows chemical structures of major species in polysorbates.Polysorbates are mainly composed of fatty acid esters sharing a commonsorbitan POE, isosorbide POE or POE head group, with oleic acid as themain fatty acid for PS80. The right panel A shows a total ion current(TIC) chromatogram of PS80 in mAb formulation by online 2D-LC/MSanalysis. The identity of the labeled peaks are: (1) POE-POEisosorbide-POE sorbitan, (2) POE sorbitan monolinoleate, (3) POEsorbitan monooleate, (4) POE isosorbide monooleate and POE monooleate,(5) POE sorbitan linoleate/oleate diester, (6) POE sorbitan di-oleate,(7) POE isosorbide di-oleate and POE di-oleate, (8) Probably POEisosorbide/POE linoleate/oleate diester as mass spectra are toocomplicated to interpret, (9) POE sorbitan mixed trioleate andtetraoleate. The right panel B shows a CAD chromatogram showing theseparation and detection of PS80 in mAb formulation by online 2D-LC/CADanalysis.

FIG. 2 shows a chromatogram of 0.1% PS80 in 50 mg/mL mAb-1 incubated at5° C. in 10 mM histidine, pH 6 for 0 hours and 36 hours according to anexemplary embodiment. Peaks eluted between 11 to 17.5 minutes were POE,POE isosorbide and POE sorbitan.

FIG. 3 shows a chart of the percentage of PS80 remaining plotted againstincubation time, where the original mAb-1, mAb-1 mixed with 0.125 μM,0.5 μM and 2 μM FP probe are indicated by filled circle with black solidline, filled diamond with red dotted line, filled square with orangedashed line and filled triangle with blue dotted line.

FIG. 4 shows a schematic diagram of the lipase(s) depletion experimentaccording to an exemplary embodiment. Streptavidin dynabeads magneticbeads were coupled with desthiobiotin-FP probe and used for lipase(s)depletion. The original mAb-1 and flow through mAb-1 as well as processcontrol mAb-1 were incubated with 0.1% PS80 at 5° C. for 36 hours andsubjected to PS degradation measurement. The enriched lipase(s) aresubjected to digestion and HCP analysis using mass spectrometry.

FIG. 5 shows a chart of percentage of PS80 remaining in original mAb-1,process control and lipase(s) depleted mAb-1, where the original mAb-1,process control and lipase(s) depleted mAb-1 are indicated by filleddiamond with black solid line, filled square with blue dotted line andfilled circle with orange dashed line.

FIG. 6A shows a chromatogram of 0.1% PS80 in 20 μg/mL commercial rabbitliver esterase incubated at 5° C. in 10 mM histidine, pH 6 for 0 hours,1.5 hours and 8 hours according to an exemplary embodiment.

FIG. 6B shows a chromatogram of 0.1% PS80 in 100 μg/mL commercial humanliver carboxylesterases 1 incubated at 5° C. in 10 mM histidine, pH 6for 0 hours, 5 hours and 18 hours according to an exemplary embodiment.

FIG. 6C shows a chromatogram of 0.1% PS80 in 50 mg/mL mAb-1 incubated at5° C. in 10 mM histidine, pH 6 for 0 hours, 18 hours and 36 hoursaccording to an exemplary embodiment.

FIG. 6D shows the sequence alignment of Liver Carboxylesterase B-1-like(A0A06117X9) (SEQ ID NO: 10), Liver Carboxylesterase 1-like (A0A061FE2)(SEQ ID NO: 11) and Human liver carboxylesterase (hCES-1) (SEQ ID NO:12).

DETAILED DESCRIPTION

Host cell proteins (HCPs) are a class of impurities that must be removedfrom all cell-derived protein therapeutics. The FDA does not specify amaximum acceptable level of HCP, but HCP concentrations in final drugproduct must be controlled and reproducible from batch to batch (FDA,1999). A primary safety concern relates to the possibility that HCPs cancause antigenic effects in human patients (Satish Kumar Singh, Impact ofProduct-Related Factors on Immunogenicity of Biotherapeutics, and 100JOURNALS OF PHARMACEUTICAL SCIENCES 354-387 (2011)). In addition toadverse health consequences for the patient, enzymatically active HCPscan potentially affect product quality during processing or long-termstorage (Sharon X. Gao et al., Fragmentation of a highly purifiedmonoclonal antibody attributed to residual CHO cell protease activity,108 BIOTECHNOLOGY AND BIOENGINEERING 977-982 (2010); Flavie Robert etal., Degradation of an Fc-fusion recombinant protein by host cellproteases: Identification of a CHO cathepsin D protease, 104BIOTECHNOLOGY AND BIOENGINEERING 1132-1141 (2009)). HCPs may present thegreatest risk for persisting through purification operations into thefinal drug product. During long-term storage, the critical qualityattributes of the product molecule must be maintained and degradation ofexcipients in the final product formulation must be minimized.

Several drug formulations on the market comprise polysorbate as one ofthe most commonly used nonionic surfactants in biopharmaceutical proteinformulation that can improve protein stability and protect drug productsfrom aggregation and denaturation (Sylvia Kiese et al., Shaken, NotStirred: Mechanical Stress Testing of an IgG1 Antibody, 97 JOURNAL OFPHARMACEUTICAL SCIENCES 4347-4366 (2008); Ariadna Martos et al., Trendson Analytical Characterization of Polysorbates and Their DegradationProducts in Biopharmaceutical Formulations, 106 JOURNAL OFPHARMACEUTICAL SCIENCES 1722-1735 (2017)). Polysorbate 20 (PS20) andpolysorbate 80 (PS80) are the most commonly used nonionic surfactants inbiopharmaceutical protein formulation that can improve protein stabilityand protect drug products from aggregation and denaturation. Typicalpolysorbate concentrations in drug products range can be between about0.001% to about 0.1% (w/v) to provide sufficient efforts on proteinstability.

Polysorbates, however, are liable to degradation that can driveundesired particulate formation in the formulated drug substances.Polysorbates are known to degrade in two main pathways: auto-oxidationand hydrolysis. Oxidation was found to be more likely to take place inPS80 due to the high content in unsaturated fatty acid estersubstituents, whereas in PS20, oxidation was believed to take place onether bond in polyoxyethylene chain that is not frequently observed(Oleg V. Borisov, Junyan A. Ji & Y. John Wang, Oxidative Degradation ofPolysorbate Surfactants Studied by Liquid Chromatography-MassSpectrometry, 104 JOURNAL OF PHARMACEUTICAL SCIENCES 1005-1018 (2015);Anthony Tomlinson et al., Polysorbate 20 Degradation inBiopharmaceutical Formulations: Quantification of Free Fatty Acids,Characterization of Particulates, and Insights into the DegradationMechanism, 12 MOLECULAR PHARMACEUTICS 3805-3815 (2015); Jia Yao et al.,A Quantitative Kinetic Study of Polysorbate Autoxidation: The Role ofUnsaturated Fatty Acid Ester Substituents, 26 PHARMACEUTICAL RESEARCH2303-2313 (2009)). In addition, polysorbates can also undergo hydrolysisby breaking the fatty acid ester bond. The particulates originating ondegradation of polysorbates can form visible or even sub-visible whichcan raise the potential for immunogenicity in patients and may havevarying effects on the drug product quality. One such possible impuritycould be fatty acid particles that are formed during manufacture,shipment, storage, handling or administration of drug formulationscomprising polysorbate. The fatty acid particles could potentially causeadverse immunogenic effects and impact shelf life. Additionally, thedegradation of polysorbates can also cause reduction in the total amountof surfactant in the formulation affecting the product's stabilityduring its manufacturing, storage, handling, and administration.

Typically, polysorbate degradation can only be observed in drug productsafter a fairly long storage time. However, PS80 degradation was observedin case of one monoclonal antibody (mAb) within 24 hours at 4° C.although no obviously high concentrated lipase was detected, suggestingunfamiliar lipase(s) existed in this drug substance. It is imperative todetect and reduce concentration(s) of such lipase(s) in order tomaintain the stability of the drug formulation.

Putative phospholipase B-like 2 (PLBD2) was the first host cell proteinthat was proposed to cause an enzymatic hydrolysis of PS20 (Nitin Dixitet al., Residual Host Cell Protein Promotes Polysorbate 20 Degradationin a Sulfatase Drug Product Leading to Free Fatty Acid Particles, 105JOURNAL OF PHARMACEUTICAL SCIENCES 1657-1666 (2016)). Porcine liveresterase was reported to be able to specifically hydrolysis ofpolysorbate 80 (not PS20) and lead the formation of PS85 over time inmAb drug product (Steven R. Labrenz, Ester Hydrolysis of Polysorbate 80in mAb Drug Product: Evidence in Support of the Hypothesized Risk Afterthe Observation of Visible Particulate in mAb Formulations, 103 JOURNALOF PHARMACEUTICAL SCIENCES 2268-2277 (2014)). Group XV lysosomalphospholipase A2 isomer X1 (LPLA₂) demonstrated the ability to degradePS20 and PS80 at less than 1 ppm (Troii Hall et al., Polysorbates 20 and80 Degradation by Group XV Lysosomal Phospholipase A 2 Isomer XI inMonoclonal Antibody Formulations, 105 JOURNAL OF PHARMACEUTICAL SCIENCES1633-1642 (2016) and Ying Cheng et al., A Rapid High-SensitivityReversed-Phase Ultra High Performance Liquid Chromatography MassSpectrometry Method for Assessing Polysorbate 20 Degradation in ProteinTherapeutics, 108 JOURNAL OF PHARMACEUTICAL SCIENCES 2880-2886 (2019)).

Recently, a range of carboxyesters, including Pseudomonas cepacia lipaseon immobead 150 (PCL), Candida antarctica lipase B on immobead 150(CALB), Thermomyces lanuginosus lipase on immobead 150 (TLL), rabbitliver esterase (RLE), Candida antarctica lipase B (CALB) and porcinepancreatic lipase type II (PPL), were selected to study the hydrolysisof two unique PS20 and PS80 which contained 99% of laurate and 98%oleate esters, respectively. Different carboxyesters showed their uniquedegradation patterns, indicating that degradation pattern can be used todifferentiate enzymes that hydrolyze polysorbates (A. C. Mcshan et al.,Hydrolysis of Polysorbate 20 and 80 by a Range of CarboxylesterHydrolases, 70 PDA JOURNAL OF PHARMACEUTICAL SCIENCE AND TECHNOLOGY332-345 (2016)). It can be essential to evaluate the effect of ahost-cell protein co-purified with a drug product on polysorbates toensure stability of the drug formulation. This can requireidentification of the host-cell protein and its ability to degradepolysorbates. Identification of host-cell proteins can be particularlychallenging since the presence of HCPs is generally in ppm range, whichmakes the isolation and identification of the HCP difficult.

The present invention discloses improved compositions comprisingpolysorbate with reduced level of host-cell proteins that can degradepolysorbate(s), methods for detection of such host-cell proteins andmethods for depleting such host-cell proteins.

Unless described otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing, particular methods and materials arenow described. All publications mentioned are hereby incorporated byreference.

The term “a” should be understood to mean “at least one”; and the terms“about” and “approximately” should be understood to permit standardvariation as would be understood by those of ordinary skill in the art;and where ranges are provided, endpoints are included.

In some exemplary embodiments, the disclosure provides a compositioncomprising a protein of interest, polysorbate, and a residual amount ofa lipase.

As used herein, the term “composition” refers to an activepharmaceutical agent that is formulated together with one or morepharmaceutically acceptable vehicles.

As used herein, the term “an active pharmaceutical agent” can include abiologically active component of a drug product. An activepharmaceutical agent can refer to any substance or combination ofsubstances used in a drug product, intended to furnish pharmacologicalactivity or to otherwise have direct effect in the diagnosis, cure,mitigation, treatment or prevention of disease, or to have direct effectin restoring, correcting or modifying physiological functions inanimals. Non-limiting methods to prepare an active pharmaceutical agentcan include using fermentation process, recombinant DNA, isolation andrecovery from natural resources, chemical synthesis, or combinationsthereof.

In some exemplary embodiments, the amount of active pharmaceutical agentin the formulation can range from about 0.01 mg/mL to about 600 mg/mL.In some specific embodiments, the amount of active pharmaceutical agentin the formulation can be about 0.01 mg/mL, about 0.02 mg/mL, about 0.03mg/mL, about 0.04 mg/mL, about 0.05 mg/mL, about 0.06 mg/mL, about 0.07mg/mL, about 0.08 mg/mL, about 0.09 mg/mL, about 0.1 mg/mL, about 0.2mg/mL, about 0.3 mg/mL, about 0.4 mg/mL, about 0.5 mg/mL, about 0.6mg/mL, about 0.7 mg/mL, about 0.8 mg/mL, about 0.9 mg/mL, about 1 mg/mL,about 2 mg/mL, about 3 mg/mL, about 4 mg/mL, about 5 mg/mL, about 6mg/mL, about 7 mg/mL, about 8 mg/mL, about 9 mg/mL, about 10 mg/mL,about 15 mg/mL, about 20 mg/mL, about 25 mg/mL, about 30 mg/mL, about 35mg/mL, about 40 mg/mL, about 45 mg/mL, about 50 mg/mL, about 55 mg/mL,about 60 mg/mL, about 65 mg/mL, about 70 mg/mL, about 5 mg/mL, about 80mg/mL, about 85 mg/mL, about 90 mg/mL, about 100 mg/mL, about 110 mg/mL,about 120 mg/mL, about 130 mg/mL, about 140 mg/mL, about 150 mg/mL,about 160 mg/mL, about 170 mg/mL, about 180 mg/mL, about 190 mg/mL,about 200 mg/mL, about 225 mg/mL, about 250 mg/mL, about 275 mg/mL,about 300 mg/mL, about 325 mg/mL, about 350 mg/mL, about 375 mg/mL,about 400 mg/mL, about 425 mg/mL, about 450 mg/mL, about 475 mg/mL,about 500 mg/mL, about 525 mg/mL, about 550 mg/mL, about 575 mg/mL, orabout 600 mg/mL.

In some exemplary embodiments, pH of the composition can be greater thanabout 5.0. In one exemplary embodiment, the pH can be greater than about5.0, greater than about 5.5, greater than about 6, greater than about6.5, greater than about 7, greater than about 7.5, greater than about 8,or greater than about 8.5.

In some exemplary embodiments, the active pharmaceutical agent can be aprotein of interest.

As used herein, the term “protein” or “protein of interest” can includeany amino acid polymer having covalently linked amide bonds. Proteinscomprise one or more amino acid polymer chains, generally known in theart as “polypeptides.” “Polypeptide” refers to a polymer composed ofamino acid residues, related naturally occurring structural variants,and synthetic non-naturally occurring analogs thereof linked via peptidebonds, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. “Synthetic peptides orpolypeptides’ refers to a non-naturally occurring peptide orpolypeptide. Synthetic peptides or polypeptides can be synthesized, forexample, using an automated polypeptide synthesizer. Various solid phasepeptide synthesis methods are known to those of skill in the art. Aprotein may contain one or multiple polypeptides to form a singlefunctioning biomolecule. A protein can include any of bio-therapeuticproteins, recombinant proteins used in research or therapy, trapproteins and other chimeric receptor Fc-fusion proteins, chimericproteins, antibodies, monoclonal antibodies, polyclonal antibodies,human antibodies, and bispecific antibodies. Another exemplary aspect, aprotein can include antibody fragments, nanobodies, recombinant antibodychimeras, cytokines, chemokines, peptide hormones, and the like.Proteins may be produced using recombinant cell-based productionsystems, such as the insect bacculovirus system, yeast systems (e.g.,Pichia sp.), mammalian systems (e.g., CHO cells and CHO derivatives likeCHO-K1 cells). For a recent review discussing biotherapeutic proteinsand their production, see Ghaderi et al., “Production platforms forbiotherapeutic glycoproteins. Occurrence, impact, and challenges ofnon-human sialylation,” (Darius Ghaderi et al., Production platforms forbiotherapeutic glycoproteins. Occurrence, impact, and challenges ofnon-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERINGREVIEWS147-176 (2012)). In some embodiments, proteins comprisemodifications, adducts, and other covalently linked moieties. Thesemodifications, adducts and moieties include for example avidin,streptavidin, biotin molecule, a modified biotin molecule, glycans(e.g., N-acetylgalactosamine, galactose, neuraminic acid,N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG,polyhistidine, FLAGtag, maltose binding protein (MBP), chitin bindingprotein (CBP), glutathione-S-transferase (GST) myc-epitope, fluorescentlabels and other dyes, and the like. Proteins can be classified on thebasis of compositions and solubility and can thus include simpleproteins, such as, globular proteins and fibrous proteins; conjugatedproteins, such as, nucleoproteins, glycoproteins, mucoproteins,chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; andderived proteins, such as, primary derived proteins and secondaryderived proteins.

In some exemplary embodiments, the protein of interest can be anantibody, a bispecific antibody, a multispecific antibody, antibodyfragment, monoclonal antibody, fusion protein, and combinations thereof.

In a particular aspect, the protein of interest can aflibercept (see,U.S. Pat. No. 7,279,159, the entire teaching of which is incorporatedherein by reference).

The term “antibody,” as used herein includes immunoglobulin moleculescomprising four polypeptide chains, two heavy (H) chains and two light(L) chains inter-connected by disulfide bonds, as well as multimersthereof (e.g., IgM). Each heavy chain comprises a heavy chain variableregion (abbreviated herein as HCVR or V_(H)) and a heavy chain constantregion. The heavy chain constant region comprises three domains, C_(H)1,C_(H)2 and C_(H)3. Each light chain comprises a light chain variableregion (abbreviated herein as LCVR or V_(L)) and a light chain constantregion. The light chain constant region comprises one domain (C_(L)1).The V_(H) and V_(L) regions can be further subdivided into regions ofhypervariability, termed complementarity determining regions (CDRs),interspersed with regions that are more conserved, termed frameworkregions (FR). Each V_(H) and V_(L) is composed of three CDRs and fourFRs, arranged from amino-terminus to carboxy-terminus in the followingorder: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. In differentembodiments of the invention, the FRs of the anti-big-ET-1 antibody (orantigen-binding portion thereof) may be identical to the human germlinesequences or may be naturally or artificially modified. An amino acidconsensus sequence may be defined based on a side-by-side analysis oftwo or more CDRs.

The term “antibody,” as used herein, also includes antigen-bindingfragments of full antibody molecules. The terms “antigen-bindingportion” of an antibody, “antigen-binding fragment” of an antibody, andthe like, as used herein, include any naturally occurring, enzymaticallyobtainable, synthetic, or genetically engineered polypeptide orglycoprotein that specifically binds an antigen to form a complex.Antigen-binding fragments of an antibody may be derived, e.g., from fullantibody molecules using any suitable standard techniques such asproteolytic digestion or recombinant genetic engineering techniquesinvolving the manipulation and expression of DNA encoding antibodyvariable and optionally constant domains. Such DNA is known and/or isreadily available from, for example, commercial sources, DNA libraries(including, e.g., phage-antibody libraries), or can be synthesized. TheDNA may be sequenced and manipulated chemically or by using molecularbiology techniques, for example, to arrange one or more variable and/orconstant domains into a suitable configuration, or to introduce codons,create cysteine residues, modify, add or delete amino acids, etc.

As used herein, an “antibody fragment” includes a portion of an intactantibody, such as, for example, the antigen-binding or variable regionof an antibody. Examples of antibody fragments include, but are notlimited to, a Fab fragment, a Fab′ fragment, a F(ab′)2 fragment, a scFvfragment, a Fv fragment, a dsFv diabody, a dAb fragment, a Fd′ fragment,a Fd fragment, and an isolated complementarity determining region (CDR)region, as well as triabodies, tetrabodies, linear antibodies,single-chain antibody molecules, and multi specific antibodies formedfrom antibody fragments. Fv fragments are the combination of thevariable regions of the immunoglobulin heavy and light chains, and ScFvproteins are recombinant single chain polypeptide molecules in whichimmunoglobulin light and heavy chain variable regions are connected by apeptide linker. In some exemplary embodiments, an antibody fragmentcontains sufficient amino acid sequence of the parent antibody of whichit is a fragment that it binds to the same antigen as does the parentantibody; in some exemplary embodiments, a fragment binds to the antigenwith a comparable affinity to that of the parent antibody and/orcompetes with the parent antibody for binding to the antigen. Anantibody fragment may be produced by any means. For example, an antibodyfragment may be enzymatically or chemically produced by fragmentation ofan intact antibody and/or it may be recombinantly produced from a geneencoding the partial antibody sequence. Alternatively, or additionally,an antibody fragment may be wholly or partially synthetically produced.An antibody fragment may optionally comprise a single chain antibodyfragment. Alternatively, or additionally, an antibody fragment maycomprise multiple chains that are linked together, for example, bydisulfide linkages. An antibody fragment may optionally comprise amulti-molecular complex. A functional antibody fragment typicallycomprises at least about 50 amino acids and more typically comprises atleast about 200 amino acids.

The phrase “bispecific antibody” includes an antibody capable ofselectively binding two or more epitopes. Bispecific antibodiesgenerally comprise two different heavy chains, with each heavy chainspecifically binding a different epitope—either on two differentmolecules (e.g., antigens) or on the same molecule (e.g., on the sameantigen). If a bispecific antibody is capable of selectively binding twodifferent epitopes (a first epitope and a second epitope), the affinityof the first heavy chain for the first epitope will generally be atleast one to two or three or four orders of magnitude lower than theaffinity of the first heavy chain for the second epitope, and viceversa. The epitopes recognized by the bispecific antibody can be on thesame or a different target (e.g., on the same or a different protein).Bispecific antibodies can be made, for example, by combining heavychains that recognize different epitopes of the same antigen. Forexample, nucleic acid sequences encoding heavy chain variable sequencesthat recognize different epitopes of the same antigen can be fused tonucleic acid sequences encoding different heavy chain constant regions,and such sequences can be expressed in a cell that expresses animmunoglobulin light chain.

A typical bispecific antibody has two heavy chains each having threeheavy chain CDRs, followed by a C_(H)1 domain, a hinge, a C_(H)2 domain,and a C_(H)3 domain, and an immunoglobulin light chain that either doesnot confer antigen-binding specificity but that can associate with eachheavy chain, or that can associate with each heavy chain and that canbind one or more of the epitopes bound by the heavy chainantigen-binding regions, or that can associate with each heavy chain andenable binding or one or both of the heavy chains to one or bothepitopes. BsAbs can be divided into two major classes, those bearing anFc region (IgG-like) and those lacking an Fc region, the latter normallybeing smaller than the IgG and IgG-like bispecific molecules comprisingan Fc. The IgG-like bsAbs can have different formats, such as, but notlimited to triomab, knobs into holes IgG (kih IgG), crossMab, orth-FabIgG, Dual-variable domains Ig (DVD-Ig), Two-in-one or dual action Fab(DAF), IgG-single-chain Fv (IgG-scFv), or κλ-bodies. The non-IgG-likedifferent formats include Tandem scFvs, Diabody format, Single-chaindiabody, tandem diabodies (TandAbs), Dual-affinity retargeting molecule(DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock(DNL) method (Gaowei Fan, Zujian Wang & minutes gju Hao, Bispecificantibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY130; Dafne Müller & Roland E. Kontermann, Bispecific Antibodies,HANDBOOK OF THERAPEUTIC ANTIBODIES265-310 (2014)).

The methods of producing BsAbs are not limited to quadroma technologybased on the somatic fusion of two different hybridoma cell lines,chemical conjugation, which involves chemical cross-linkers, and geneticapproaches utilizing recombinant DNA technology. Examples of bsAbsinclude those disclosed in the following patent applications, which arehereby incorporated by reference: U.S. Ser. No. 12/823,838, filed Jun.25, 2010; U.S. Ser. No. 13/488628, filed Jun. 5, 2012; U.S. Ser. No.14/031,075, filed Sep. 19, 2013; U.S. Ser. No. 14/808,171, filed Jul.24, 2015; U.S. Ser. No. 15/713,574, filed Sep. 22, 2017; U.S. Ser. No.15/713,569, field Sep. 22, 2017; U.S. Ser. No. 15/386,453, filed Dec.21, 2016; U.S. Ser. No. 15/386,443, filed Dec. 21, 2016; U.S. Ser. No.15/22343 filed Jul. 29, 2016; and U.S. Ser. No. 15/814,095, filed Nov.15, 2017. Low levels of homodimer impurities can be present at severalsteps during the manufacturing of bispecific antibodies. The detectionof such homodimer impurities can be challenging when performed usingintact mass analysis due to low abundances of the homodimer impuritiesand the co-elution of these impurities with main species when carriedout using a regular liquid chromatographic method.

As used herein “multispecific antibody” or “Mab” refers to an antibodywith binding specificities for at least two different antigens. Whilesuch molecules normally will only bind two antigens (i.e., bispecificantibodies, BsAbs), antibodies with additional specificities such astrispecific antibody and KIH Trispecific can also be addressed by thesystem and method disclosed herein.

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. A monoclonal antibodycan be derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, by any means available or known in the art.Monoclonal antibodies useful with the present disclosure can be preparedusing a wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof.

In some exemplary embodiments, the protein of interest can have a pI inthe range of about 4.5 to about 9.0. In one exemplary specificembodiment, the pI can be about 4.5, about 5.0, about 5.5, about 5.6,about 5.7, about 5.8, about 5.9, about 6.0, about 6.1 about 6.2, about6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9,about 7.0, about 7.1 about 7.2, about 7.3, about 7.4, about 7.5, about7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1 about 8.2,about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about8.9, or about 9.0.

In some exemplary embodiments, the types of protein of interest in thecompositions can be at least two. In some specific embodiments, one ofthe at least two protein of interest can be a monoclonal antibody, apolyclonal antibody, a bispecific antibody, an antibody fragment, afusion protein, or an antibody-drug complex. In some other specificembodiments, concentration of one of the at least two protein ofinterest can be about 20 mg/mL to about 400 mg/mL. In some exemplaryembodiments, the types of protein of interest in the compositions aretwo. In some exemplary embodiments, the types of protein of interest inthe compositions are three. In some exemplary embodiments, the types ofprotein of interest in the compositions are five.

In some exemplary embodiments, the two or more protein of interest inthe composition can be selected from trap proteins, chimeric receptorFc-fusion proteins, chimeric proteins, antibodies, monoclonalantibodies, polyclonal antibodies, human antibodies, bispecificantibodies, multispecific antibodies, antibody fragments, nanobodies,recombinant antibody chimeras, cytokines, chemokines, or peptidehormones.

In some exemplary embodiments, the composition can be a co-formulation.

In some exemplary embodiments, the protein of interest can be purifiedfrom mammalian cells. The mammalian cells can be of human origin ornon-human origin can include primary epithelial cells (e.g.,keratinocytes, cervical epithelial cells, bronchial epithelial cells,tracheal epithelial cells, kidney epithelial cells and retinalepithelial cells), established cell lines and their strains (e.g., 293embryonic kidney cells, BHK cells, HeLa cervical epithelial cells andPER-C6 retinal cells, MDBK (NBL-1) cells, 911 cells, CRFK cells, MDCKcells, CHO cells, BeWo cells, Chang cells, Detroit 562 cells, HeLa 229cells, HeLa S3 cells, Hep-2 cells, KB cells, LSI80 cells, LS174T cells,NCI-H-548 cells, RPMI2650 cells, SW-13 cells, T24 cells, WI-28 VA13, 2RAcells, WISH cells, BS—C—I cells, LLC-MK2 cells, Clone M-3 cells, 1-10cells, RAG cells, TCMK-1 cells, Y-1 cells, LLC-PKi cells, PK(15) cells,GHi cells, GH3 cells, L2 cells, LLC-RC 256 cells, MHiCi cells, XC cells,MDOK cells, VSW cells, and TH-I, B1 cells, BSC-1 cells, RAf cells,RK-cells, PK-15 cells or derivatives thereof), fibroblast cells from anytissue or organ (including but not limited to heart, liver, kidney,colon, intestines, esophagus, stomach, neural tissue (brain, spinalcord), lung, vascular tissue (artery, vein, capillary), lymphoid tissue(lymph gland, adenoid, tonsil, bone marrow, and blood), spleen, andfibroblast and fibroblast-like cell lines (e.g., CHO cells, TRG-2 cells,IMR-33 cells, Don cells, GHK-21 cells, citrullinemia cells, Dempseycells, Detroit 551 cells, Detroit 510 cells, Detroit 525 cells, Detroit529 cells, Detroit 532 cells, Detroit 539 cells, Detroit 548 cells,Detroit 573 cells, HEL 299 cells, IMR-90 cells, MRC-5 cells, WI-38cells, WI-26 cells, Midi cells, CHO cells, CV-1 cells, COS-1 cells,COS-3 cells, COS-7 cells, Vero cells, DBS-FrhL-2 cells, BALB/3T3 cells,F9 cells, SV-T2 cells, M-MSV-BALB/3T3 cells, K-BALB cells, BLO-11 cells,NOR-10 cells, C3H/IOTI/2 cells, HSDMiC3 cells, KLN205 cells, McCoycells, Mouse L cells, Strain 2071 (Mouse L) cells, L-M strain (Mouse L)cells, L-MTK′ (Mouse L) cells, NCTC clones 2472 and 2555, SCC-PSA1cells, Swiss/3T3 cells, Indian muntjac cells, SIRC cells, Cn cells, andJensen cells, Sp2/0, NS0, NS1 cells or derivatives thereof).

In some exemplary embodiments, the composition can be stable. Thestability of a composition can comprise evaluating the chemicalstability, physical stability or functional stability of the activepharmaceutical agent. The formulations of the present inventiontypically exhibit high levels of stability of the active pharmaceuticalagent.

In terms of protein formulations, the term “stable,” as used hereinrefers to the protein of interest within the formulations being able toretain an acceptable degree of chemical structure or biological functionafter storage under exemplary conditions defined herein. A formulationmay be stable even though the protein of interest contained therein doesnot maintain 100% of its chemical structure or biological function afterstorage for a defined amount of time. Under certain circumstances,maintenance of about 90%, about 95%, about 96%, about 97%, about 98% orabout 99% of a protein's structure or function after storage for adefined amount of time may be regarded as “stable”.

Stability can be measured, inter alia, by determining the percentage ofnative protein(s) that remain in the formulation after storage for adefined amount of time at a defined temperature. The percentage ofnative protein can be determined by, inter alia, size exclusionchromatography (e.g., size exclusion high performance liquidchromatography [SE-HPLC]), such that native means non-aggregated andnon-degraded. An “acceptable degree of stability,” as that phrase isused herein, means that at least 90% of the native form of the proteincan be detected in the formulation after storage for a defined amount oftime at a given temperature. In certain embodiments, at least about 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the native formof the protein can be detected in the formulation after storage for adefined amount of time at a defined temperature. The defined amount oftime after which stability is measured can be at least 14 days, at least28 days, at least 1 month, at least 2 months, at least 3 months, atleast 4 months, at least 5 months, at least 6 months, at least 7 months,at least 8 months, at least 9 months, at least 10 months, at least 11months, at least 12 months, at least 18 months, at least 24 months, ormore.

Stability can be measured, inter alia, by determining the percentage ofprotein that forms in an aggregate within the formulation after storagefor a defined amount of time at a defined temperature, wherein stabilityis inversely proportional to the percent aggregate that is formed. Thisform of stability is also referred to as “colloidal stability” herein.The percentage of aggregated protein can be determined by, inter alia,size exclusion chromatography (e.g., size exclusion high performanceliquid chromatography [SE-HPLC]). An “acceptable degree of stability,”as that phrase is used herein, means that at most 6% of the protein isin an aggregated form detected in the formulation after storage for adefined amount of time at a given temperature. In certain embodiments anacceptable degree of stability means that at most about 6%, 5%, 4%, 3%,2%, 1%, 0.5%, or 0.1% of the protein can be detected in an aggregate inthe formulation after storage for a defined amount of time at a giventemperature. The defined amount of time after which stability ismeasured can be about at least 2 weeks, at least 28 days, at least 1month, at least 2 months, at least 3 months, at least 4 months, at least5 months, at least 6 months, at least 7 months, at least 8 months, atleast 9 months, at least 10 months, at least 11 months, at least 12months, at least 18 months, at least 24 months, or more. The temperatureat which the pharmaceutical formulation may be stored when assessingstability can be any temperature from about −80° C. to about 45° C.,e.g., storage at about −80° C., about −30° C., about −20° C., about 0°C., about 4° C., about 5° C., about 25° C., about 35° C., about 37° C.or about 45° C. For example, a pharmaceutical formulation may be deemedstable if after six months of storage at 5° C., less than about 3%, 2%,1%, 0.5%, or 0.1% of the protein is detected in an aggregated form. Apharmaceutical formulation may also be deemed stable if after six monthsof storage at about 25° C., less than about 4%, 3%, 2%, 1%, 0.5%, or0.1% of the protein is detected in an aggregated form. A pharmaceuticalformulation may also be deemed stable if after 28 days of storage at 45°C., less than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the proteinis detected in an aggregated form. A pharmaceutical formulation may alsobe deemed stable if after three months of storage at −20° C., −30° C.,or −80° C. less than about 3%, 2%, 1%, 0.5%, or 0.1% of the protein isdetected in an aggregated form.

Stability can also be measured, inter alia, by determining thepercentage of protein that forms in an aggregate within the formulationafter storage for a defined amount of time at a defined temperature,wherein stability is inversely proportional to the percent aggregatethat is formed. This form of stability is also referred to as “colloidalstability” herein. The percentage of aggregated protein can bedetermined by, inter alia, size exclusion chromatography (e.g., sizeexclusion high performance liquid chromatography [SE-HPLC]). Anacceptable degree of stability,” as that phrase is used herein, meansthat at most about 6% of the protein is in an aggregated form detectedin the formulation after storage for a defined amount of time at a giventemperature. In certain embodiments an acceptable degree of stabilitymeans that at most about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of theprotein can be detected in an aggregate in the formulation after storagefor a defined amount of time at a given temperature. The defined amountof time after which stability is measured can be about at least 2 weeks,at least 28 days, at least 1 month, at least 2 months, at least 3months, at least 4 months, at least 5 months, at least 6 months, atleast 7 months, at least 8 months, at least 9 months, at least 10months, at least 11 months, at least 12 months, at least 18 months, atleast 24 months, or more. The temperature at which the pharmaceuticalformulation may be stored when assessing stability can be anytemperature from about −80° C. to about 45° C., for example, storage atabout −80° C., about −30° C., about −20° C., about 0° C., about 4°−8°C., about 5° C., about 25° C., about 35° C., about 37° C. or about 45°C. For example, a pharmaceutical formulation may be deemed stable ifafter six months of storage at about 5° C., less than about 3%, 2%, 1%,0.5%, or 0.1% of the protein is detected in an aggregated form. Apharmaceutical formulation may also be deemed stable if after six monthsof storage at about 25° C., less than about 4%, 3%, 2%, 1%, 0.5%, or0.1% of the protein is detected in an aggregated form. A pharmaceuticalformulation may also be deemed stable if after about 28 days of storageat 45° C., less than about 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of theprotein is detected in an aggregated form. A pharmaceutical formulationmay also be deemed stable if after three months of storage at about −20°C., −30° C., or −80° C. less than about 3%, 2%, 1%, 0.5%, or 0.1% of theprotein is detected in an aggregated form.

Stability can be also measured, inter alia, by determining thepercentage of protein that migrates in a more acidic fraction during ionexchange (“acidic form”) than in the main fraction of protein (“maincharge form”), wherein stability is inversely proportional to thefraction of protein in the acidic form. While not wishing to be bound bytheory, deamidation of the protein may cause the protein to become morenegatively charged and thus more acidic relative to the non-deamidatedprotein (see, e.g., Robinson, N. (2002) “Protein Deamidation” PNAS,99(8):5283-5288). The percentage of “acidified” protein can bedetermined by, inter alia, ion exchange chromatography (e.g., cationexchange high performance liquid chromatography [CEX-HPLC]). An“acceptable degree of stability,” as that phrase is used herein, meansthat at most 49% of the protein is in a more acidic form detected in theformulation after storage for a defined amount of time at a definedtemperature. In certain exemplary embodiments, an acceptable degree ofstability means that at most about 49%, 45%, 40%, 35%, 30%, 25%, 20%,15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the protein can bedetected in an acidic form in the formulation after storage for adefined amount of time at a given temperature. The defined amount oftime after which stability is measured can be about at least 2 weeks, atleast 28 days, at least 1 month, at least 2 months, at least 3 months,at least 4 months, at least 5 months, at least 6 months, at least 7months, at least 8 months, at least 9 months, at least 10 months, atleast 11 months, at least 12 months, at least 18 months, at least 24months, or more.

The temperature at which the pharmaceutical formulation may be storedwhen assessing stability can be any temperature from about −80° C. toabout 45° C., e.g., storage at about −80° C., about −30° C., about −20°C., about 0° C., about 4°−8° C., about 5° C., about 25° C., or about 45°C. For example, a pharmaceutical formulation may be deemed stable ifafter three months of storage at −80° C., −30° C., or −20° C. less thanabout 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%,17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%,0.5% or 0.1% of the protein is in a more acidic form. A pharmaceuticalformulation may also be deemed stable if after six months of storage at5° C., less than about 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%,22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the protein is in a more acidicform. A pharmaceutical formulation may also be deemed stable if aftersix months of storage at 25° C., less than about 43%, 42%, 41%, 40%,39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%,25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%,10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the protein isin a more acidic form. A pharmaceutical formulation may also be deemedstable if after 28 days of storage at 45° C., less than about 49%, 48%,47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%,33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%,19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%,2%, 1%, 0.5% or 0.1% of the protein can be detected in a more acidicform.

Other methods may be used to assess the stability of the formulations ofthe present invention such as, for example differential scanningcalorimetry (DSC) to determine thermal stability, controlled agitationto determine mechanical stability, and absorbance at about 350 nm orabout 405 nm to determine solution turbidities. For example, aformulation of the present invention may be considered stable if, after6 or more months of storage at about 5° C. to about 25° C., the changein OD405 of the formulation is less than about 0.05 (e.g., 0.04, 0.03,0.02, 0.01, or less) from the OD405 of the formulation at time zero.Measuring the biological activity or binding affinity of the protein toits target may also be used to assess stability. For example, aformulation of the present invention may be regarded as stable if, afterstorage at e.g., 5° C., 25° C., 45° C., etc. for a defined amount oftime (e.g., 1 to 12 months), the protein contained within theformulation binds to its target with an affinity that is at least 90%,95%, or more of the binding affinity of the protein prior to saidstorage. Binding affinity may be determined by e.g., ELISA or plasmonresonance. Biological activity may be determined by a protein activityassay, such as for example, contacting a cell that expresses the proteinwith the formulation comprising the protein. The binding of the proteinto such a cell may be measured directly, such as, for example, via FACSanalysis. Alternatively, the downstream activity of the protein systemmay be measured in the presence of the protein and compared to theactivity of the protein system in the absence of protein.

In some exemplary embodiments, the composition can be used for thetreatment, prevention and/or amelioration of a disease or disorder.Exemplary, non-limiting diseases and disorders that can be treatedand/or prevented by the administration of the pharmaceuticalformulations of the present invention include, infections; respiratorydiseases; pain resulting from any condition associated with neurogenic,neuropathic or nociceptic pain; genetic disorder; congenital disorder;cancer; herpetiformis; chronic idiopathic urticarial; scleroderma,hypertrophic scarring; Whipple's Disease; benign prostate hyperplasia;lung disorders, such as mild, moderate or severe asthma, allergicreactions; Kawasaki disease, sickle cell disease; Churg-Strausssyndrome; Grave's disease; pre-eclampsia; Sjogren's syndrome; autoimmunelymphoproliferative syndrome; autoimmune hemolytic anemia; Barrett'sesophagus; autoimmune uveitis; tuberculosis; nephrosis; arthritis,including chronic rheumatoid arthritis; inflammatory bowel diseases,including Crohn's disease and ulcerative colitis; systemic lupuserythematosus; inflammatory diseases; HIV infection; AIDS; LDLapheresis; disorders due to PCSK9-activating mutations (gain of functionmutations, “GOF”), disorders due to heterozygous FamilialHypercholesterolemia (heFH); primary hypercholesterolemia; dyslipidemia;cholestatic liver diseases; nephrotic syndrome; hypothyroidism; obesity;atherosclerosis; cardiovascular diseases; neurodegenerative diseases;neonatal Onset Multisystem Inflammatory Disorder (NOM ID/CINCA);Muckle-Wells Syndrome (MWS); Familial Cold Autoinflammatory Syndrome(FCAS); familial Mediterranean fever (FMF); tumor necrosis factorreceptor-associated periodic fever syndrome (TRAPS); systemic onsetjuvenile idiopathic arthritis (Still's Disease); diabetes mellitus type1 and type 2; auto-immune diseases; motor neuron disease; eye diseases;sexually transmitted diseases; tuberculosis; disease or condition whichis ameliorated, inhibited, or reduced by a VEGF antagonist; disease orcondition which is ameliorated, inhibited, or reduced by a PD-1inhibitor; disease or condition which is ameliorated, inhibited, orreduced by a Interleukin antibody; disease or condition which isameliorated, inhibited, or reduced by a NGF antibody; disease orcondition which is ameliorated, inhibited, or reduced by a PCSK9antibody; disease or condition which is ameliorated, inhibited, orreduced by a ANGPTL antibody; disease or condition which is ameliorated,inhibited, or reduced by an activin antibody; disease or condition whichis ameliorated, inhibited, or reduced by a GDF antibody; disease orcondition which is ameliorated, inhibited, or reduced by a Fel d 1antibody; disease or condition which is ameliorated, inhibited, orreduced by a CD antibody; disease or condition which is ameliorated,inhibited, or reduced by a C5 antibody or combinations thereof.

In some exemplary embodiments, the composition can be administered to apatient. Administration may be via any route acceptable to those skilledin the art. Non-limiting routes of administration include oral, topical,or parenteral. Administration via certain parenteral routes may involveintroducing the formulations of the present invention into the body of apatient through a needle or a catheter, propelled by a sterile syringeor some other mechanical device such as a continuous infusion system. Acomposition provided by the present invention may be administered usinga syringe, injector, pump, or any other device recognized in the art forparenteral administration. A composition of the present invention mayalso be administered as an aerosol for absorption in the lung or nasalcavity. The compositions may also be administered for absorption throughthe mucus membranes, such as in buccal administration.

As used herein, “polysorbate” refers to a common excipient used informulation development to protect antibodies against various physicalstresses such as agitation, freeze-thaw processes, and air/waterinterfaces (Emily Ha, Wei Wang & Y. John Wang, Peroxide formation inpolysorbate 80 and protein stability, 91 JOURNAL OF PHARMACEUTICALSCIENCES 2252-2264 (2002); Bruce A. Kerwin, Polysorbates 20 and 80 Usedin the Formulation of Protein Biotherapeutics: Structure and DegradationPathways, 97 JOURNAL OF PHARMACEUTICAL SCIENCES 2924-2935 (2008);Hanns-Christian Mahler et al., Adsorption Behavior of a Surfactant and aMonoclonal Antibody to Sterilizing-Grade Filters, 99 Journal ofPharmaceutical Sciences 2620-2627 (2010)) and can include a non-ionic,amphipathic surfactant composed of fatty acid esters ofpolyoxyethylene-sorbitan. The esters can include polyoxyethylenesorbitan head group and either a saturated monolaurate side chain(polysorbate 20; PS20) or an unsaturated monooleate side chain(polysorbate 80; PS80). In some exemplary embodiments, the polysorbatecan be present in the formulation in the range of 0.001% to 2%(weight/volume). Polysorbate can also contain a mixture of various fattyacid chains; for example, polysorbate 80 contains oleic, palmitic,myristic and stearic fatty acids, with the monooleate fraction making upapproximately 58% of the polydisperse mixture (Nitin Dixit et al.,Residual Host Cell Protein Promotes Polysorbate 20 Degradation in aSulfatase Drug Product Leading to Free Fatty Acid Particles, 105 JOURNALOF PHARMACEUTICAL SCIENCES 1657-1666 (2016)). Non-limiting examples ofpolysorbates include polysorbate-20, polysorbate-40, polysorbate-60,polysorbate-65, and polysorbate-80.

A polysorbate can be susceptible to auto-oxidation in a pH- andtemperature-dependent manner, and additionally, exposure to UV light canalso produce instability (Ravuri S.k. Kishore et al., Degradation ofPolysorbates 20 and 80: Studies on Thermal Autoxidation and Hydrolysis,100 JOURNAL OF PHARMACEUTICAL SCIENCES 721-731 (2011)), resulting infree fatty acids in solution along with the sorbitan head group. Thefree fatty acids resulting from polysorbate can include any aliphaticfatty acids with six to twenty carbons. Non-limiting examples of freefatty acids include oleic acid, palmitic acid, stearic acid, myristicacid, lauric acid, or combinations thereof.

In some exemplary embodiments, the polysorbate can form free fatty acidparticles. The free fatty acid particles can be at least 5 μm in size.Further, these fatty acid particles can be classified according to theirsize as visible (>100 μm), sub-visible (<100 which can be sub-dividedinto micron (1-100 μm) and submicron (100 nm-1000 nm)) and nanometerparticles (<100 nm) (Linda Narhi, Jeremy Schmit & Deepak Sharma,Classification of protein aggregates, 101 JOURNAL OF PHARMACEUTICALSCIENCES 493-498). In some exemplary embodiments, the fatty acidparticles can be visible particles. Visible particles can be determinedby visual inspection. In some exemplary embodiments, the fatty acidparticles can be sub-visible particles. Subvisible particles can bemonitored by the light blockage method according to United StatesPharmacopeia (USP).

In some exemplary embodiments, the concentration of polysorbate in thecomposition can be about 0.001% w/v, about 0.002% w/v, about 0.003% w/v,about 0.004% w/v, about 0.005% w/v, about 0.006% w/v, about 0.007% w/v,about 0.008% w/v, about 0.009% w/v, about 0.01% w/v, about 0.011% w/v,about 0.015% w/v, about 0.02% w/v, 0.025% w/v, about 0.03% w/v, about0.035% w/v, about 0.04% w/v, about 0.045% w/v, about 0.05% w/v, about0.055% w/v, about 0.06% w/v, about 0.065% w/v, about 0.07% w/v, about0.075% w/v, about 0.08% w/v, about 0.085% w/v, about 0.09% w/v, about0.095% w/v, about 0.1% w/v, about 0.11% w/v, about 0.115% w/v, about0.12% w/v, about 0.125% w/v, about 0.13% w/v, about 0.135% w/v, about0.14% w/v, about 0.145% w/v, about 0.15% w/v, about 0.155% w/v, about0.16% w/v, about 0.165% w/v, about 0.17% w/v, about 0.175% w/v, about0.18% w/v, about 0.185% w/v, about 0.19% w/v, about 0.195% w/v, or about0.2% w/v.

In some exemplary embodiments, the polysorbate can be degraded by thelipase(s) present in the composition. These lipase(s) can be aprocess-related impurity which can be derived from the manufacturingprocess and can include the three major categories: cellsubstrate-derived, cell culture-derived and downstream derived. Cellsubstrate-derived impurities include, but are not limited to, proteinsderived from the host organism and nucleic acid (host cell genomic,vector, or total DNA). Cell culture-derived impurities include, but arenot limited to, inducers, antibiotics, serum, and other mediacomponents. Downstream-derived impurities include, but are not limitedto, enzymes, chemical and biochemical processing reagents (e.g.,cyanogen bromide, guanidine, oxidizing and reducing agents), inorganicsalts (e.g., heavy metals, arsenic, nonmetallic ion), solvents,carriers, ligands (e.g., monoclonal antibodies), and other leachables.

In one aspect, the lipase can be a serine hydrolase. In a specificaspect, then lipase can be carboxylesterase B-1-like protein(A0A061I7X9). In another specific aspect, the lipase can be livercarboxylesterase 1-like protein (A0A061IFE2). In yet another specificaspect, the lipase can be both carboxylesterase B-1-like protein andliver carboxylesterase 1-like protein.

The effect of lipases on degradation of polysorbate was identified byusing detecting methods according to some exemplary embodiments.

Having identified lipases that can degrade polysorbates in certainprotein preparations, it would be highly advantageous and desirable tohave reagents, methods, and kits for the specific, sensitive, andquantitative determination and/or depletion of such lipase levels, aswell as to develop methods of preparing compositions with low levels oflipases.

In some exemplary embodiments, the disclosure provides compositionswhich comprises less than about 5 ppm of carboxylesterase B-1-likeprotein and/or liver carboxylesterase 1-like protein.

In some exemplary embodiments, the residual amount of carboxylesteraseB-1-like protein and/or liver carboxylesterase 1-like protein in thecomposition can be less than about 5 ppm. In some specific exemplaryembodiments, the residual amount of carboxylesterase B-1-like proteinand/or liver carboxylesterase 1-like protein is less than about 0.01ppm, about 0.02 ppm, about 0.03 ppm, about 0.04 ppm, about 0.05 ppm,about 0.06 ppm, 0.07 ppm, 0.08 ppm, 0.09 ppm, about 0.1 ppm, about 0.2ppm, about 0.3 ppm, about 0.4 ppm, about 0.5 ppm, about 0.6 ppm, 0.7ppm, 0.8 ppm, 0.9 ppm, about 1 ppm, about 2 ppm, about 3 ppm, about 4ppm, or about 5 ppm.

In some exemplary embodiments, the disclosure provides various methodsof preparing a composition having a protein of interest which comprisesless than about 5 ppm of carboxylesterase B-1-like protein and/or livercarboxylesterase 1-like protein.

The disclosure also provides a method of preparing a composition havinga protein of interest with less than about 5 ppm of carboxylesteraseB-1-like protein and/or liver carboxylesterase 1-like protein comprisingforming a sample with the protein of interest and the lipase, contactingthe sample with a probe, said probe capable of binding to the lipase toform a complex and separating the complex from the sample.

In some exemplary embodiments, the sample can be obtained from any stepof the bioprocess, such as, culture cell culture fluid (CCF), harvestedcell culture fluid (HCCF), process performance qualification (PPQ), anystep in the downstream processing, drug solution (DS), or a drug product(DP) comprising the final formulated product. In some other specificexemplary embodiments, the sample can be selected from any step of thedownstream process of clarification, chromatographic purification, viralinactivation, or filtration. In some specific exemplary embodiments, thedrug product can be selected from manufactured drug product in theclinic, shipping, storage, or handling. In some other specific exemplaryembodiments, the drug product can comprise polysorbate(s).

In some exemplary embodiments, the method of preparing a compositionhaving a protein of interest with less than about 5 ppm ofcarboxylesterase B-1-like protein and/or liver carboxylesterase 1-likeprotein can also include further chromatographic steps.

In some exemplary embodiments, method of preparing a composition havinga protein of interest with less than about 5 ppm of carboxylesteraseB-1-like protein and/or liver carboxylesterase 1-like protein canfurther include filtering one or all of the following: sample, eluatefrom one or more of the chromatographic steps, and/or flow-through fromone or more of the chromatographic steps.

As used herein, “viral filtration” can include filtration using suitablefilters including, but not limited to, Planova 20N™, 50 N or BioEx fromAsahi Kasei Pharma, Viresolve™ filters from EMD Millipore, ViroSart CPVfrom Sartorius, or Ultipor DV20 or DV50™ filter from Pall Corporation.It will be apparent to one of ordinary skill in the art to select asuitable filter to obtain desired filtration performance.

In some exemplary embodiments, method of preparing a composition havinga protein of interest with less than about 5 ppm of carboxylesteraseB-1-like protein and/or liver carboxylesterase 1-like protein canfurther include performing UF/DF on one or all of the following: sample,eluate from one or more of the chromatographic steps, and/orflow-through from one or more of the chromatographic steps.

As used herein, the term “ultrafiltration” or “UF” can include amembrane filtration process similar to reverse osmosis, usinghydrostatic pressure to force water through a semi-permeable membrane.Ultrafiltration is described in detail in: LEOS J. ZEMAN & ANDREW L.ZYDNEY, MICROFILTRATION AND ULTRAFILTRATION: PRINCIPLES AND APPLICATIONS(1996). Filters with a pore size of smaller than 0.1 μm can be used forultrafiltration. By employing filters having such small pore size, thevolume of the sample can be reduced through permeation of the samplebuffer through the filter while antibodies are retained behind thefilter.

As used herein, “diafiltration” or “DF” can include a method of usingultrafilters to remove and exchange salts, sugars, and non-aqueoussolvents, to separate free from bound species, to remove lowmolecular-weight material, and/or to cause the rapid change of ionicand/or pH environments. Microsolutes are removed most efficiently byadding solvent to the solution being ultrafiltered at a rateapproximately equal to the ultrafiltration rate. This washesmicrospecies from the solution at a constant volume, effectivelymanufacturing the retained antibody. In certain embodiments of thepresent invention, a diafiltration step can be employed to exchange thevarious buffers used in connection with the instant invention,optionally prior to further chromatography or other purification steps,as well as to remove impurities from the antibody preparation.

In some exemplary embodiments, the probe can be capable of being linkedon a solid support. The solid support may be any of the well knownsupports or matrices which are currently widely used or proposed forimmobilisation, separation etc. These may take the form of particles,sheets, gels, filters, membranes, fibres, capillaries, or microtitrestrips, tubes, plates or wells etc. Conveniently the support may be madeof glass, silica, latex or a polymeric material. Particulate materials,for example, beads are generally preferred due to their greater bindingcapacity, particularly polymeric beads. A particulate solid support usedaccording to the invention will comprise spherical beads. Non-magneticpolymer beads suitable for use in the method of the invention areavailable from Dyno Particles AS (Lillestrom, Norway) as well as fromQiagen, Pharmacia and Serotec.

However, to aid manipulation and separation, magnetic beads arepreferred. The term “magnetic” as used herein means that the support iscapable of having a magnetic moment imparted to it when placed in amagnetic field, and thus is displaceable under the action of that field.In other words, a support comprising magnetic particles may readily beremoved by magnetic aggregation, which provides a quick, simple andefficient way of separating the particles following the nucleic acidbinding step, and is a far less rigorous method than traditionaltechniques such as centrifugation which generate shear forces which maydegrade nucleic acids. Thus, using the method of the invention, thecomplex formed between the probe and lipase may be removed byapplication of a magnetic field, for example, using a permanent magnet.It is usually sufficient to apply a magnet to the side of the vesselcontaining the sample mixture to aggregate the particles to the wall ofthe vessel and to pour away the remainder of the sample. In somespecific aspects, the superparamagnetic particles can be used, forexample those described by Sintef in EP-A-106873, as magneticaggregation and clumping of the particles during reaction can beavoided, thus ensuring uniform and nucleic acid extraction. Thewell-known magnetic particles sold by Dynal AS (Oslo, Norway) asDYNABEADS, are particularly suited to use in the present invention.Further, beads, or other supports, may be prepared having differenttypes of functionalised surface, for example positively charged orhydrophobic. Weakly and strongly positively charged surfaces, weaklynegatively charged neutral surfaces and hydrophobic surfaces e.g.polyurethane-coated have been shown to work well.

In some exemplary embodiments, the probe can be capable of being linkedon a solid support using a ligand. Non-limiting examples can include anindicator, biotin molecule, a modified biotin molecule, modified biotinmolecule, a modified biotin molecule, a nuclei, a protein sequence, anepitope tag, an electron poor molecule or an electron rich molecule.Specific examples of ligands can include, but are not limited to, biotinmolecule or a modified such as deiminobiotin molecule, desthiobiotinmolecule, vicinal diols, such as 1,2-dihydroxyethane,1,2-dihydroxycyclohexane, etc., digoxigenin, maltose, oligohistidine,glutathione, 2,4-dintrobenzene, phenylarsenate, ssDNA, dsDNA, a peptideof polypeptide, a metal chelate, a saccharide, rhodamine or fluorescein,or any hapten to which an antibody can be generated. Examples of ligandsand their capture reagents include but are not limited to: dethiobiotinor structurally modified biotin-based reagents, including deiminobiotinmolecule, a modified biotin molecule, which bind to proteins of theavidin/streptavidin family, which may, for example, be used in the formsof strepavidin-Agarose, oligomeric-avidin-Agarose, ormonomeric-avidin-Agarose; any 1,2-diol, such as 1,2-dihydroxyethane(HO—CH₂—CH₂—OH), and other 1,2-dihyroxyalkanes including those of cyclicalkanes, for example, 1,2-dihydroxycyclohexane which bind to an alkyl oraryl boronic acid or boronic acid esters, such as phenyl-B(OH)₂ orhexyl-B(OEthyl)₂ which may be attached via the alkyl or aryl group to asolid support material, such as Agarose; maltose which binds to maltosebinding protein (as well as any other sugar/sugar binding protein pairor more generally to any ligand/ligand binding protein pairs that hasproperties discussed above); a hapten, such as the dinitrophenyl group,for any antibody where the hapten binds to an anti-hapten antibody thatrecognizes the hapten, for example the dinitrophenyl group will bind toan anti-dinitrophenyl-1gG; a ligand which binds to a transition metal,for example, an oligomeric histidine will bind to Ni(II), the transitionmetal capture reagent may be used in the form of a resin bound chelatedtransition metal, such as nitrilotriacetic acid-chelated Ni(II) oriminodiacetic acid-chelated Ni(II); glutathione which binds toglutathione-S-transferase.

The disclosure also provides a method of detecting a livercarboxylesterase-1-like protein or liver carboxylesterase-B1-likeprotein in a sample by contacting the sample with a serine hydrolaseprobe. In one aspect, the method of detecting a lipase in a sample cancomprise contacting and incubating the sample with a serine hydrolaseprobe to form a complex of lipase and serine hydrolase probe. In afurther aspect, the method of detecting a lipase in a sample cancomprise filtering out the serine hydrolase probe that does not form thecomplex of lipase and serine hydrolase probe.

In some exemplary embodiments, the method of detecting a lipase in asample can further comprise contacting the contacting the sample withmagnetic beads having an ability to bind to the serine hydrolase probesuch that magnetic beads are bound to the complex of lipase and serinehydrolase probe.

In some specific exemplary embodiments, the magnetic beads bound to thecomplex of lipase and serine hydrolase probe can be further removed fromthe sample and washed with a buffer.

In some exemplary embodiments, the method of detecting a lipase in asample can further comprise removing the magnetic beads, which are boundto the complex of lipase and serine hydrolase probe to form a solutionof enriched lipase.

In some exemplary embodiments, the method of detecting a lipase in asample can further comprise adding hydrolyzing agent to the solution toobtain digests.

As used herein, the term “hydrolyzing agent” refers to any one orcombination of a large number of different agents that can performdigestion of a protein. Non-limiting examples of hydrolyzing agents thatcan carry out enzymatic digestion include protease from AspergillusSaitoi, elastase, subtilisin, protease XIII, pepsin, trypsin, Tryp-N,chymotrypsin, aspergillopepsin I, LysN protease (Lys-N), LysCendoproteinase (Lys-C), endoproteinase Asp-N(Asp-N), endoproteinaseArg-C(Arg-C), endoproteinase Glu-C(Glu-C) or outer membrane protein T(OmpT), immunoglobulin-degrading enzyme of Streptococcus pyogenes(IdeS), thermolysin, papain, pronase, V8 protease or biologically activefragments or homologs thereof or combinations thereof. Non-limitingexamples of hydrolyzing agents that can carry out non-enzymaticdigestion include the use of high temperature, microwave, ultrasound,high pressure, infrared, solvents (non-limiting examples are ethanol andacetonitrile), immobilized enzyme digestion (IMER), magnetic particleimmobilized enzymes, and on-chip immobilized enzymes. For a recentreview discussing the available techniques for protein digestion seeSwitazar et al., “Protein Digestion: An Overview of the AvailableTechniques and Recent Developments” (Linda Switzar, Martin Giera &Wilfried M. A. Niessen, Protein Digestion: An Overview of the AvailableTechniques and Recent Developments, 12 JOURNAL OF PROTEOME RESEARCH1067-1077 (2013)). One or a combination of hydrolyzing agents can cleavepeptide bonds in a protein or polypeptide, in a sequence-specificmanner, generating a predictable collection of shorter peptides.

The ratio of hydrolyzing agent to the lipase and the time required fordigestion can be appropriately selected to obtain a digestion of thelipase. When the enzyme to substrate ratio is unsuitably high, thecorrespondingly high digestion rate will not allow sufficient time forthe peptides to be analyzed by mass spectrometer, and sequence coveragewill be compromised. On the other hand, a low E/S ratio would need longdigestion and thus long data acquisition time. The enzyme to substrateratio can range from about 1:0.5 to about 1:200. As used herein, theterm “digestion” refers to hydrolysis of one or more peptide bonds of aprotein. There are several approaches to carrying out digestion of aprotein in a sample using an appropriate hydrolyzing agent, for example,enzymatic digestion or non-enzymatic digestion.

In some exemplary embodiments, the method of detecting a lipase in asample can further comprise adding protein denaturing agent to thesolution.

As used herein, “protein denaturing” can refer to a process in which thethree-dimensional shape of a molecule is changed from its native statewithout rupture of peptide bonds. The protein denaturation can becarried out using a protein denaturing agent. Non-limiting examples of aprotein denaturing agent include heat, high or low pH, or exposure tochaotropic agents. Several chaotropic agents can be used as proteindenaturing agents. Chaotropic solutes increase the entropy of the systemby interfering with intramolecular interactions mediated by non-covalentforces such as hydrogen bonds, van der Waals forces, and hydrophobiceffects. Non-limiting examples for chaotropic agents include butanol,ethanol, guanidinium chloride, lithium perchlorate, lithium acetate,magnesium chloride, phenol, propanol, sodium dodecyl sulfate, thiourea,N-lauroylsarcosine, urea, and salts thereof. In a specific aspect, theprotein denaturing agent can be urea.

In some exemplary embodiments, the method of detecting a lipase in asample can further comprise adding protein denaturing or reducing agentto the solution.

As used herein, the term “protein reducing agent” refers to the agentused for reduction of disulfide bridges in a protein. Non-limitingexamples of the protein reducing agents used to reduce the protein aredithiothreitol (DTT), ß-mercaptoethanol, Ellman's reagent, hydroxylaminehydrochloride, sodium cyanoborohydride, tris(2-carboxyethyl)phosphinehydrochloride (TCEP-HCl), or combinations thereof. In one aspect, theprotein reducing agent can be DTT (dithiothreitol).

In some exemplary embodiments, the method of detecting a lipase in asample can further comprise adding protein alkylating agent to thesolution.

As used herein, the term “protein alkylating agent” refers to the agentused for alkylation certain free amino acid residues in a protein.Non-limiting examples of the protein alkylating agents are iodoacetamide(I0A), chloroacetamide (CAA), acrylamide (AA), N-ethylmaleimide (NEM),methyl methanethiosulfonate (MMTS), and 4-vinylpyridine or combinationsthereof.

In some exemplary embodiments, the method of detecting a lipase in asample can further comprise analyzing the digests to detect the lipases.In one aspect, the digests can be analyzed using a mass spectrometer. Ina specific aspect, the mass spectrometer can be a tandem massspectrometer. In another specific aspect, the mass spectrometer can becoupled to a liquid chromatography system. In yet another specificaspect, the mass spectrometer can be coupled to a liquidchromatography—multiple reaction monitoring system.

As used herein, the term “mass spectrometer” includes a device capableof identifying specific molecular species and measuring their accuratemasses. The term is meant to include any molecular detector into which apolypeptide or peptide may be eluted for detection and/orcharacterization. A mass spectrometer can include three major parts: theion source, the mass analyzer, and the detector. The role of the ionsource is to create gas phase ions. Analyte atoms, molecules, orclusters can be transferred into gas phase and ionized eitherconcurrently (as in electrospray ionization) or through separateprocesses. The choice of ion source depends heavily on the application.

As used herein, the term “tandem mass spectrometry” includes a techniquewhere structural information on sample molecules is obtained by usingmultiple stages of mass selection and mass separation. A prerequisite isthat the sample molecules can be transferred into gas phase and ionizedintact and that they can be induced to fall apart in some predictableand controllable fashion after the first mass selection step. MultistageMS/MS, or MSn, can be performed by first selecting and isolating aprecursor ion (MS²), fragmenting it, isolating a primary fragment ion(MS³), fragmenting it, isolating a secondary fragment (MS⁴), and so onas long as one can obtain meaningful information, or the fragment ionsignal is detectable. Tandem MS has been successfully performed with awide variety of analyzer combinations. What analyzers to combine for acertain application can be determined by many different factors, such assensitivity, selectivity, and speed, but also size, cost, andavailability. The two major categories of tandem MS methods aretandem-in-space and tandem-in-time, but there are also hybrids wheretandem-in-time analyzers are coupled in space or with tandem-in-spaceanalyzers. A tandem-in-space mass spectrometer comprises an ion source,a precursor ion activation device, and at least two non-trapping massanalyzers. Specific m/z separation functions can be designed so that inone section of the instrument ions are selected, dissociated in anintermediate region, and the product ions are then transmitted toanother analyzer for m/z separation and data acquisition. Intandem-in-time, mass spectrometer ions produced in the ion source can betrapped, isolated, fragmented, and m/z separated in the same physicaldevice.

The peptides identified by the mass spectrometer can be used assurrogate representatives of the intact protein and their posttranslational modifications. They can be used for proteincharacterization by correlating experimental and theoretical MS/MS data,the latter generated from possible peptides in a protein sequencedatabase. The characterization includes, but is not limited, tosequencing amino acids of the protein fragments, determining proteinsequencing, determining protein de novo sequencing, locatingpost-translational modifications, or identifying post translationalmodifications, or comparability analysis, or combinations thereof.

As used herein, the term “database” refers to bioinformatic tools whichprovide the possibility of searching the uninterpreted MS-MS spectraagainst all possible sequences in the database(s). Non-limiting examplesof such tools are Mascot (http://www.matrixscience.com), Spectrum Mill(http://www.chem.agilent.com), PLGS (http://www.waters.com), PEAKS(http://www.bioinformaticssolutions.com), Proteinpilot(http://download.appliedbiosystems.com//proteinpilot), Phenyx(http://www.phenyx-ms.com), Sorcerer (http://www.sagenresearch.com),OMSSA (http://www.pubchem.ncbi.nlm.nih.gov/omssa/), X!Tandem(http://www.thegpm.org/TANDEM/), Protein Prospector (http://www.http://prospector.ucsf.edu/prospector/mshome.htm), Byonic(https://www.proteinmetrics.com/products/byonic) or Sequest(http://fieldsserippsedu/sequest).

In some exemplary embodiments, the mass spectrometer can be coupled to aliquid chromatography system.

As used herein, the term “chromatography” refers to a process in which achemical mixture carried by a liquid or gas can be separated intocomponents as a result of differential distribution of the chemicalentities as they flow around or over a stationary liquid or solid phase.Non-limiting examples of chromatography include traditionalreversed-phased (RP), ion exchange (IEX) and normal phase chromatography(NP). Unlike RP, NP and IEX chromatography, in which hydrophobicinteraction, hydrophilic interaction and ionic interaction respectivelyare the dominant interaction modes, mixed-mode chromatography can employa combination of two or more of these interaction modes. Several typesof liquid chromatography can be used with the mass spectrometer, suchas, rapid resolution liquid chromatography (RRLC), ultra-performanceliquid chromatography (UPLC), ultra-fast liquid chromatography (UFLC)and nano liquid chromatography (nLC). For further details onchromatography method and principles, see Colin et al. (CoLIN F. POOLEET AL., LIQUID CHROMATOGRAPHY FUNDAMENTALS AND INSTRUMENTATION (2017)).

In some exemplary embodiments, the mass spectrometer can be coupled to anano liquid chromatography. In some exemplary embodiments, the mobilephase used to elute the protein in liquid chromatography can be a mobilephase that can be compatible with a mass spectrometer.

In some specific exemplary embodiments, the mobile phase can be ammoniumacetate, ammonium bicarbonate, or ammonium formate, or combinationsthereof.

In some exemplary embodiments, the mass spectrometer can be coupled to aliquid chromatography—multiple reaction monitoring system.

As used herein, “multiple reaction monitoring” or “MRM” refers to a massspectrometry-based technique that can precisely quantify smallmolecules, peptides, and proteins within complex matrices with highsensitivity, specificity and a wide dynamic range (Paola Picotti & RuediAebersold, Selected reaction monitoring-based proteomics: workflows,potential, pitfalls and future directions, 9 NATURE METHODS 555-566(2012)). MRM can be typically performed with triple quadrupole massspectrometers wherein a precursor ion corresponding to the selectedsmall molecules/peptides is selected in the first quadrupole and afragment ion of the precursor ion was selected for monitoring in thethird quadrupole (Yong Seok Choi et al., Targeted human cerebrospinalfluid proteomics for the validation of multiple Alzheimers diseasebiomarker candidates, 930 JOURNAL OF CHROMATOGRAPHY B129-135 (2013)).

In some exemplary embodiments, the mass spectrometer can be coupled to aliquid chromatography—selected reaction monitoring system.

It is understood that the present invention is not limited to any of theaforesaid, chromatographic resin(s), excipient(s), filtration method(s),hydrolyzing agent(s), protein denaturing agent(s), protein alkylatingagent(s), instrument(s) used for identification, and any chromatographicresin(s), excipient(s), filtration method(s), hydrolyzing agent(s),protein denaturing agent(s), protein alkylating agent(s), instrument(s)used for identification can be selected by any suitable means.

Various publications, including patents, patent applications, publishedpatent applications, accession numbers, technical articles and scholarlyarticles are cited throughout the specification. Each of these citedreferences is incorporated by reference herein in its entirety and forall purposes.

The present invention will be more fully understood by reference to thefollowing Examples. They should not, however, be construed as limitingthe scope of the invention

EXAMPLES Materials.

Dynabeads MyOne Streptavidin T1 was purchased from Invitrogen of ThermoFisher Scientific (Waltham, Mass.). ActivX Desthiobiotin-FP SerineHydrolase Probe, Formic acid, acetonitrile, Diothiothreitol (DTT) and1-step ultra TMD-blotting solution were purchased from Thermo FisherScientific (Waltham, Mass.). Acetic acid, 10X Tris buffered saline(TBS), Iodoacetamide (IAM), bovine serum albumin (BSA) and urea werepurchased from Sigma-Aldrich (St. Louis, Mo.). HEPES buffered salinewith EDTA and 0.005% v/v Surfactant P-20 (HBS-EP) was purchased from GE(Boston, Mass.). Monoclonal antibody drug substance was made atRegeneron Pharmaceutical Inc. Polysorbate 80 were purchased from Croda(East Yorkshire, UK). Rabbit rLE was purchased from Sigma Aldrich (St.Louis, Mo.). Human CES-1 was purchased from Abcam (Cambridge UK).Sequencing Grade Modified Trypsin was purchased from Promega (Madison,Wis.). Oasis Max column (2.1×20 mm, 30 μm) and Acquity UPLC BEH C4column (2.1×50 mm, 1.7 μm) were purchased from Waters (Milford, Mass.).Acclaim PepMap 100 C18 analytical column (0.075×250 mm, 3 μm) andAcclaim PepMap 100 C18 trap column (0.075×20 mm, 3 μm) were purchasedfrom Thermo Fisher Scientific (Waltham, Mass.). DPBS (10x) was purchasedfrom Gibco (Thermo Fisher Scientific, Waltham, Mass.) and Tween20 waspurchased from J. T. Baker (Phillipsburg, N.J.). Q-Exactive Plus withelectrospray ionization (ESI) source was purchased from Thermo FisherScientific (Waltham, Mass.).

Two-Dimensional Liquid Chromatography-Charged Aerosol Detection(CAD)/Mass Spectrometry (MS) Method to Analyze Polysorbate Degradation.

The degradation of PS20 and PS80 in CHO cell-free media or formulatedantibody were analyzed by two-dimensional HPLC-CAD/MS method aspreviously described by Genentech (Yi Li et al., Characterization andStability Study of Polysorbate 20 in Therapeutic Monoclonal AntibodyFormulation by Multidimensional Ultrahigh-Performance LiquidChromatography-Charged Aerosol Detection-Mass Spectrometry, 86ANALYTICAL CHEMISTRY 5150-5157 (2014)). Polysorbates were firstseparated from formulated mAb using Oasis MAX column (2.1×20 mm, 30 μm)pre-equilibrated with 99% solvent A (0.1% formic acid in water) and 1%solvent B (0.1% formic acid in acetonitrile). Post sample injection, theequilibration gradient was held for 1 minute, followed by a linearincrease of Solvent B to 15% in 4 minutes to separate polysorbate frommAb. The eluted polysorbates were then diverted to Acquity BEH C4 column(2.1×50 mm, 1.7 μm) using a switch valve for reversed phasechromatography-based separation. At the start of the separation, solventB was quickly increased to 20% in 1.5 minutes, then gradually increasedto 99% at 45 minutes and held for 5 minutes, followed by anequilibration step of 1% B for 5 minutes. The flow rate was kept at 0.1mL/min and column temperature at 40° C.

The 2D-LC system was set up with Thermo UltiMate 3000 and coupled withCorona Ultra CAD detector, operating at nitrogen pressure of 75 psi forquantitation. Chromeleon 7 was used for system control and dataanalysis. Q-Exactive Plus with ESI source was coupled with the 2D-LCsystem for characterization only. The instrument was operated in apositive mode with capillary voltage at 3.8 kV, capillary temperature at350° C., sheath flow rate at 40, and aux flow rate at 10. Full scanspectra were collected over the m/z range of 150-2000. Thermo Xcalibursoftware was used to collect and analyze MS data.

Peak area of each ester was obtained from the CAD chromatogram and addedup to account for intact PS80. The remaining percentage of PS80 afterdegradation was calculated by comparing the sum of the peak area ofmonoester eluting between 25 minutes and 30 minutes at each time pointto the sum of peak areas at time zero. Relative percentage of differentorder ester or total esters can be calculated similarly.

PS80 Degradation Assay with Human CES-1, Rabbit LES and FormulatedAntibody.

The effect of human CES-1 and rabbit LES on PS80 was examined by mixing2 μL 0.1 mg/mL human CES-1 or 0.02 mg/mL rabbit LES with 2 μL 1% PS80 in16 μL 10 mM histidine buffer, pH 6.0, followed with incubation at 4° C.for 1.5, 8 and 18 hours, respectively. One aliquot (3 μL) of eachsolution was diluted 25 times by adding 72 μL 10 mM histidine, pH 6.0,before the LC-CAD analysis.

The hydrolysis of PS80 in formulated mAb was examined by mixing 18 μL 50mg/mL mAb (buffer exchange to 10 mM histidine, pH 6.0) with 2 μL 1% PS80then incubated at 5° C. for 18, 24 and 36 hours. One aliquot (3 μL) ofeach solution was diluted 25 times with 10 mM histidine, pH 6.0 beforethe LC-CAD analysis.

Inhibition of Lipases from CHO-Derived Antibodies.

ActivX Desthiobiotin-FP serine hydrolase probe were diluted in DMSO to0.1 mM as stock solution. Aliquoted 1.25 μL, 5 μL and 20 μL probe stocksolution each was mixed with 5 mg mAb in 1 X PBS in a final volume of 1mL, followed by gentle rotating at room temperature for 1 hour. Eachmixture was then buffer exchanged into 10 mM histidine, pH 6.0 to removethe free probes, and the mAb concentration was adjusted to 50 mg/mL.Each buffer exchanged sample was incubated with 0.1% PS80 at 5° C.followed by LC-CAD PS80 degradation assay.

Depletion of Lipases from CHO-Derived Antibodies.

Lipases depletion experiment was performed by using immobilized ActivXDesthiobiotin-FP serine hydrolase probe. To immobilize the probe, 35 μLActivX Desthiobiotin-FP serine hydrolase probe (0.1 mM stock solution inDMSO) was first coupled with 2 mg Streptavidin Dynabeads to a finalvolume of 1 mL in 1 X PBS by gentle rotating at room temperature for 2hours. Process control sample was prepared by mixing 35 μL DMSO with 2mg Streptavidin Dynabeads to a final volume of 1 mL in 1 X and gentlerotating at room temperature for 2 hours. The beads were washed by 1 XPBS 3 times and then resuspended into 800 μL 1 X PBS. 5 mg mAb samplewas then added into the FP probe-coupled Streptavidin Dynabeads andincubated at room temperature with gentle rotation for 1 hour. Thesupernatant was buffer exchanged into 10 mM histidine, pH 6.0, and themAb concentration adjusted to 50 mg/mL. The buffer-exchanged supernatantsamples were then incubated with 0.1% PS80 at 5° C. followed by LC-CADPS80 degradation assay.

Detection of Host Cell Proteins (HCPs) in CHO-Derived Antibodies withABPP.

ActivX Desthiobiotin-FP serine hydrolase probe were diluted in DMSO to0.1 mM as stock solution. Aliquoted 20 μL probe stock solution was firstmixed with 5 mg mAb in 1 X PBS to a final volume of 1 mL, followed bygentle rotating at room temperature for 1 hour. Free probes were removedby filtration and protein was recovered by 5 M urea in PBS. 2 mgStreptavidin Dynabeads was added to the solution and incubated by gentlerotating at room temperature for 2 hours. After removing thesupernatant, Dynabeads were collected by magnet, and washed by 5 M ureain PBS and then resuspended into 5 M urea/50 mM tris solution with 5 mMTCEP. The proteins were denatured and reduced at 55° C. for 30 minutesand then incubated with 10 mM iodoacetamide for 30 minutes in dark.Alkylated proteins were diluted 5 times and digested with 1 μg trypsinat 37° C. for overnight. Dynabeads were removed by magnet and thesupernatant with peptide mixture was acidified by 5 μL of 10% FA,desalted using GL-Tip™ SDB desalting tip (GL science, Japan) andresuspended into 40 μL 0.1% FA. 15 μL were transferred to Eppendorftubes for Nano LC-MS/MS analysis and the rest were stored at −80° C.Negative control was performed by heating mAb sample at 80° C. for 5minutes first to denature all proteins to prevent host cell proteinsfrom binding to the ActivX Desthiobiotin-FP serine hydrolase probe.

LC-MS/MS Analysis.

The peptide mixture was dissolved in 40 μL of 0.1% formic acid (FA) and10 μL was first loaded onto a 20 cm×0.075 mm Acclaim PepMap 100 C18 trapcolumn (Thermo Fisher Scientific) for desalting and later separated on a250 mm×0.075 mm Acclaim PepMap 100 C18 analytical column in an UltiMate3000 nanoLC (Thermo Fisher Scientific). The mobile phase A was made of0.1% FA in ultra-pure water and mobile phase B was made of 0.1% FA in80% ACN. The peptides were separated with a 150 minute linear gradientof 2%-32% of buffer B at flow rate of 300 nL/min. The UltiMate 3000nanoLC was coupled with a Q-Exactive HFX mass spectrometer (ThermoFisher Scientific). The mass spectrometer was operated in thedata-dependent mode in which the 10 most intense ions were subjected tohigher-energy collisional dissociation (HCD) fragmentation with thenormalized collision energy (NCE) 27%, AGC 3e6, max injection time 60 msfor each full MS scan (from m/z 375-1500 with resolution of 120,000) andAGC 1e5, max injection time 60 ms for MS/MS events (from m/z 200-2000with resolution of 30,000).

mAb-1 Direct Digestion.

100 μg of mAb-1 was dried with speed vacuum, then re-constituted with 20μL 8 M urea containing 10 mM DTT. The protein was denatured and reducedat 55° C. for 30 minutes, and then incubated with 6 μL of 50 mg/mLiodoacetamide for 30 minutes in dark. Alkylated protein was digestedwith 100 μL 0.1 μg/μL trypsin at 37° C. for overnight. The peptidemixture was acidified by 5 μL of 10% TFA. The sample was diluted to 0.4μg/μL and 2 μL was injected onto the column for LC-MS/MS analysis.

PRM analysis of CES-B1L and CES-1L in mAb-1.

Direct digestion of samples (0.8 μg) were loaded onto a 20 cm×0.075 mmAcclaim PepMap 100 C18 trap column (Thermo Fisher Scientific) fordesalting and later separated on a 250 mm×0.075 mm Acclaim PepMap 100C18 analytical column in an UltiMate 3000 nanoLC (Thermo FisherScientific). The column was preequilibrated with 98% mobile phase A(made of 0.1% formic acid in water) and 2% mobile phase B (made of 0.1%formic acid in 80% ACN) at a flow rate of 300 nL/min. Post sampleinjection a linear gradient from 2% to 37% mobile phase B was appliedover 100 minutes to separate the peptides. Mass spectrometry data wereacquired by parallel reaction monitoring (PRM) targeting 3 peptidesLNVQGDTK [m/z 437.7351²⁺](SEQ ID NO: 1), AISESGVILVPGLFTK [m/z815.9744²⁺](SEQ ID NO: 2) and ENHAFVPTVLDGVLLPK [m/z 925.0145²⁺](SEQ IDNO: 3) from CES-1L, 3 peptides APEEILAEK [m/z 500.2715²⁺](SEQ ID NO: 4),DGASEEETNLSK [m/z 640.2861²⁺](SEQ ID NO: 5) and IRDGVLDILGDLTFGIPSVIVSR[m/z 819.1355³⁺](SEQ ID NO: 6) from CES-B1L, and 3 peptides GPSVFPLAPCSR[644.3293²⁺](SEQ ID NO: 7), LLIYDASNRPTGIPAR [586.3283³⁺](SEQ ID NO: 8)and STSESTAALGCLVK [712.3585²⁺](SEQ ID NO: 9) from mAb-1. In allexperiments, a full mass spectrum at 120,000 resolution relative to m/z200 (AGC target 1e6, 60 ms maximum injection time, m/z 350-2000) wasfollowed by time scheduled PRM scans at 30,000 resolution (AGC target1e5, 100 ms maximum injection time). Higher energy collisionaldissociation (HCD) was used with 27 eV normalized collision energy andan isolation window of 2 m/z for MS/MS analysis.

EXAMPLE 1

Polysorbate in mAb Formulation Detected by 2D-LC-CAD/MS

Polysorbate in formulated mAbs was separated, identified, andquantitated by 2D-LC-CAD/MS following slightly modified method by Yi Liet al., supra and Oleg V. Borisov et al., Toward Understanding MolecularHeterogeneity of Polysorbates by Application of LiquidChromatography-Mass Spectrometry with Computer-Aided Data Analysis, 83ANALYTICAL CHEMISTRY 3934-3942 (2011). The first dimensional LC by OasisMax column was designed to remove mAb, and the second dimensionalreversed phase chromatography was implemented to separate the remainingPOE and POE esters based on their fatty acid content and type. The PS80species eluted in the order of POE, POE isosorbide, POE sorbitan,monoesters, diesters, triesters and tetraesters (FIG. 1, right panel).The structure of each ester was elucidated by mass spectrometry based onthe chemical formula of the polymer and dioxolanylium ion generated byin-source fragmentation. FIG. 1 right panel A is the representativetotal ion current (TIC) chromatogram of PS80 with major peaks labeled,in the eluting order of POE-POE isosorbide-POE sorbitan, POE sorbitanmonolinoleate, POE sorbitan monooleate, POE isosorbide monooleate andPOE monooleate, POE sorbitan linoleate/oleate diester, POE sorbitandi-oleate, POE isosorbide di-oleate and POE di-oleate, Probably POEisosorbide/POE linoleate/oleate diester and POE sorbitan mixed trioleateand tetraoleate. It should be noted that peak 8 in FIG. 1 was labeled aspossible POE isosorbide/POE linoleate/oleate diester mixer as the massspectra were too complicated to interpret. Quantitation of polysorbateswas determined by charged aerosol detection (CAD) chromatographyanalysis (FIG. 1, right panel B).

EXAMPLE 2

Rapid PS80 Degradation in mAb-1 Formulation

Rapid PS80 degradation was observed in mAb-1 during storage at 5° C. for36 hours. Significant decreases occurred in peaks eluting between 25 and30 minutes, representing POE monoesters, i.e., POE sorbitanmonolinoleate, POE sorbitan monooleate, POE isosorbide monooleate andPOE monooleate, while POEs eluting between 10 and 18 minutes showedsignificant increases (FIG. 2). There were no changes on POE di-, tri-and tetra-esters eluting between 32-45 minutes. This unique degradationpattern suggests that it is more likely one family of lipase/esteraseresponsible for PS80 degradation. This family of hydrolases can onlydegrade the monoester part of PS80 while leaving higher order estersuntouched. If more than one type of hydrolase was involved in thedegradation, the degradation pattern would be more complex.

EXAMPLE 3

Inhibition of Lipases by Desthiobiotin-Fluorophosphonate (FP) ProbeResults in a Vanished PS80 Degradation

Because most of the lipases that have been reported to degradepolysorbates belong to the family of serine hydrolases, we carried outinhibition experiments using the FP probe. This experiment enables theidentification and distinction of enzymatically active hydrolyses fromother inactive hydrolyses either in their zymogen form or withendogenous inhibitors. The rationale of this experiment is that if thereis any active serine hydrolase, adding its inhibitor would stop theenzyme from functioning, in our case, degrading PS80. Desthiobiotin-FPprobe is one of the commercially available serine hydrolase probes thatcontain the reactive fluorophosphonate group which forms covalent bondwith Ser at the catalytic center of the serine type hydrolase and blocksits enzymatic activity. The inhibition experiment clearly demonstratedthat by adding as little as 0.125 μM of the FP probe, the enzymaticactivity was completely stopped (FIG. 3). This experiment alsodemonstrated that only the serine type of lipase is presented in theformulated drug substance as the desthiobiotin-FP probe is specific toserine hydrolase.

EXAMPLE 4

Depletion of Lipases by Desthiobiotin-Fluorophosphonate (FP) ProbeResults in a Diminished PS80 Degradation in formulated mAb

We then performed depletion of lipases using immobilized ActivX FPserine hydrolase probe. The biotin part of the probe can be captured andimmobilized on Streptavidin surface, allowing enrichment andpurification of the captured serine hydrolases. The design of thedepletion experiment serves two goals: 1) if PS80 degradation is causedby lipase(s) belonging to the serine hydrolase family, depletion willresult in a diminished PS80 degradation; 2) the lipase(s) captured onthe Desthiobiotin-FP probe can be further identified by massspectrometry analysis.

The depletion experiment was performed as outlined in the Material andMethods section with depletion scheme for mAbs shown in FIG. 4.Desthiobiotin-FP probe was coupled to Streptavidin Dynabeads fordepletion of lipases. As shown in FIG. 5, prior to lipase depletion,approximately 44.7% of PS80 monoester degradation in mAb-1 was observedafter 18 h incubation at 5° C. Additional 18 h incubation led tocomplete PS80 monoesters loss. After lipase depletion, less than 8% PS80degradation was observed after either 18 h or 36 h incubation. Thedepletion results demonstrated that the lipase(s) that degraded PS80 inmAb-1 was removed by the desthiobitin-FP probe. To ensure that it is theprobe rather than streptavidin magnetic beads that interacted with thelipases, the experiments were performed by introducing a process controlsample. The process control sample was produced by mixing mAb-1 withstreptavidin magnetic beads only without adding desthiobiotin-FP probe.Approximately 29% and 86% of PS80 degradation in mAbl was observed after18 h and 36 h 5° C. incubation, respectively, indicating that there werecertain non-specific interactions between lipases and magnetic beads.Compared to the FP probe, the lipase removed by the non-specificinteractions was significantly less, therefore, the majority of thelipase was removed by specific binding between the immobilized FP probeand lipase.

EXAMPLE 5

Liver Carboxylesterases were Identified in the FP Probe-EnrichedFraction from mAb-1

The host cell proteins captured by the desthiobiotin-FP probe weresubject to tryptic digestion and mass spectrometry analysis as describedin material and method section. Among the 15 host cell proteinsidentified (Table 1), CES-B1L and CES-1L were identified for the firsttime. By comparing with the denatured control sample (Table 2), it wasconcluded that both proteins were captured by specific binding to the FPprobe. CES-1L was only identified in the active form but not in thedenatured form suggesting that it was biologically active in mAb-1.CES-B1L was identified in the active form with 13 unique peptides whileonly 2 unique peptides in the denatured forms, suggesting a small amountof CES-B1L protein was able to bind non-specifically to the magneticbeads, and the results were in line with process control results in thedepletion experiment (FIG. 5). Nevertheless, the much higher number ofunique peptides identified in the active forms suggests that CES-B1L isalso responsible for PS80 degradation. For other identified host cellproteins, most can be easily determined as non-active enzymes as theywere found in both conditions with a similar number of unique peptides,for example, cullin-9-like protein and ceruloplasmin. Anionic trypsin-2was identified in the active form of mAb-1 as it is also a serineprotease, however, it can be excluded owing to its function as aprotease irrelevant to PS80 degradation. The other two co-capturedproteins, actin and annexin, can also be excluded from degrading PS80due to their lack of enzymatic functions.

To determine the abundance of the newly identified lipases, CES-B1L andCES-1L, PRM analysis were performed. The concentrations of CES-B1L andCES-1L were determined to be 9.6 and 9.0 ppm, respectively. Therelatively low abundance of these two lipases suggests that theirenzymatic activity for degrading PS80 is strong.

TABLE 1 Enriched host cell protein by FP probe identified in nativemAb-1 # Uniq. Protein in mAb-1 (native) Accession # Peps. Livercarboxylesterase B-1-like protein A0A061I7X9 13 Liver carboxylesterase1-like protein A0A061IFE2 7 Peroxiredoxin-1 Q9JKY1 7 Cullin-9-likeprotein A0A061IMU7 7 Junction plakoglobin G3HLU9 6 Transthyretin G3I4M96 Glyceraldehyde-3-phosphate dehydrogenase A0A061IDP2 3 CeruloplasminA0A061INJ7 3 Annexin G3IG05 3 Anionic Trypsin-2 G3HL18 3 VitaminD-binding protein G3IHJ6 3 Ubiquitin-40S ribosomal protein S27a-likeA0A061IQ58 3 protein Desmocollin-2-like protein A0A061IEG0 2tyrosine-protein kinase receptor G3HG67 2 Actin, aortic smooth muscleG3HQY2 2

TABLE 2 Enriched host cell protein by FP probe identified in denaturedmAb-1 # Uniq. Protein in mAb-1 (denatured) Accession # Peps.Cullin-9-like protein A0A061IMU7 7 Transthyretin G3I4M9 4 Ubiquitin-40Sribosomal protein S27a-like A0A061IQ58 3 proteinGlyceraldehyde-3-phosphate dehydrogenase A0A061IDP2 3 Peroxiredoxin-1Q9JKY1 3 Vitamin D-binding protein G3IHJ6 3 Ceruloplasmin A0A061INJ7 2tyrosine-protein kinase receptor G3HG67 2 Desmocollin-2-like proteinA0A061IEG0 2 Liver carboxylesterase B-1-like protein A0A061I7X9 2Junction plakoglobin G3HLU9 1

EXAMPLE 6

PS80 Degradation Pattern with human Liver Carboxylesterase-1 and RabbitLiver Esterase

One common and important practice in HCP analysis is to validate thefunction of lipase activities. The inhibition and depletion experimentshave provided strong evidence that CES-B1L and CES-1L are most likelythe lipases responsible for PS80 degradation. However, considering theFP probe that was used is not specific to a single protein but to afamily of proteins, it is possible that other lipase(s) presented in thedrug product that may also play a role in the degradation pathway. Aspiked-in experiment can offer the essential verification on whether thesuspected lipases are the root cause of PS80 degradation. If thespiked-in lipase can generate exactly the same degradation pattern asthe endogenous lipase, the identified lipase can then be confirmed asthe key element for PS degradation. This confirmation is usuallydifficult due to the lack of available active lipases. To furtherconfirm the role of these two newly identified lipases, a BLAST searchwas performed. The search results suggested commercially availablerabbit liver esterase and human liver carboxylesterase 1 arefunctionally similar with each having 56.0% and 69.7% sequence homologyto the first segment of CES-B1L and CES-1L, respectively (FIG. 6D). Bothhuman liver carboxylesterase 1 and rabbit liver esterase were chosen tocompare the PS80 degradation pattern as mAb-1. FIG. 6 showed that bothhuman and rabbit liver esterase exhibited an equivalent degradationpattern as that showed in mAb-1 (FIG. 6, A-C). In all three samples, therapidly degraded components of PS80 were monoesters, which elutedbetween 25 to 30 minutes, while di-, tri- and tetra-esters which elutedafter 32 minutes remained unchanged. The signature degradation patternexperiments by the known lipases verified that CES-B1L and CES-1L wereresponsible for PS80 degradation in mAb-1.

What is claimed is:
 1. A method of depleting lipase from a sample, comprising: contacting the sample including lipase with a probe, said probe capable of binding to the lipase to form a complex; and separating the complex from the sample to thereby deplete the lipase from the sample
 2. The method of claim 1, wherein the sample comprises a protein of interest.
 3. The method of claim 1, wherein the sample comprises a polysorbate excipient.
 4. The method of claim 3, wherein the polysorbate excipient is selected from polysorbate-20, polysorbate-60, polysorbate-80 or combinations thereof.
 5. The method of claim 1, wherein the lipase is liver carboxylesterase-B1-like protein.
 6. The method of claim 1, wherein the lipase is liver carboxylesterase-1-like protein.
 7. The method of claim 1, wherein the probe is capable of being linked to a solid support.
 8. The method of claim 7, wherein the solid support is agarose beads.
 9. The method of claim 7, wherein the solid support is magnetic beads.
 10. The method of claim 1, wherein the probe is attached to a solid support using a ligand.
 11. The method of claim 10, wherein the ligand can be an indicator, biotin molecule, a modified biotin molecule, a nuclei, a sequence, an epitope tag, an electron poor molecule or an electron rich molecule.
 12. The method of claim 1 further comprising recovering the lipase from the complex.
 13. A method of a method of purifying a sample having a protein of interest and a lipase, comprising: contacting the sample with a probe, said probe capable of binding to the lipase to form a complex; and separating the complex from the sample to thereby purify the protein of interest in the sample.
 14. The method of claim 13, wherein the sample comprises a polysorbate excipient.
 15. The method of claim 14, wherein the polysorbate excipient is selected from polysorbate-20, polysorbate-60, polysorbate-80 or combinations thereof.
 16. The method of claim 13, wherein the lipase is liver carboxylesterase-B1-like protein.
 17. The method of claim 13, wherein the lipase is liver carboxylesterase-1-like protein.
 18. The method of claim 13, wherein the probe is capable of being linked to a solid support.
 19. The method of claim 18, wherein the solid support is agarose beads.
 20. The method of claim 18, wherein the solid support is magnetic beads.
 21. The method of claim 13, wherein the probe is attached to a solid support using a ligand.
 22. The method of claim 21, wherein the ligand can be an indicator, biotin molecule, a modified biotin molecule, a nuclei, a sequence, an epitope tag, an electron poor molecule or an electron rich molecule.
 23. The method of claim 13 further comprising recovering the lipase from the complex.
 24. A method of decreasing degradation of polysorbate in a sample, comprising contacting the sample including lipase and polysorbate with a probe, said probe capable of binding to the lipase to form a complex; and separating the complex from the sample to thereby decreasing degradation of polysorbate in the sample.
 25. The method of claim 24, wherein the sample further comprises a protein of interest.
 26. The method of claim 24, wherein the polysorbate is selected from polysorbate-20, polysorbate-60, polysorbate-80 or combinations thereof.
 27. The method of claim 24, wherein the lipase is liver carboxylesterase-B1-like protein.
 28. The method of claim 24, wherein the lipase is liver carboxylesterase-1-like protein.
 29. The method of claim 24, wherein the probe is capable of being linked to a solid support.
 30. The method of claim 29, wherein the solid support is agarose beads.
 31. The method of claim 29, wherein the solid support is magnetic beads.
 32. The method of claim 24, wherein the probe is attached to a solid support using a ligand.
 33. The method of claim 32, wherein the ligand can be an indicator, biotin molecule, a modified biotin molecule, a nuclei, a sequence, an epitope tag, an electron poor molecule or an electron rich molecule.
 34. The method of claim 24 further comprising recovering the lipase from the complex.
 35. A composition having a protein of interest purified from mammalian cells, surfactant and a residual amount of liver carboxylesterase B-1-like protein, wherein the residual amount of liver carboxylesterase B-1-like protein is less than about 5 ppm.
 36. The composition of claim 35, wherein the surfactant is polysorbate
 80. 37. The composition of claim 36, wherein the liver carboxylesterase B-1-like protein causes degradation of the polysorbate
 80. 38. The composition of claim 35, wherein the composition is a parenteral formulation
 39. The composition of claim 36, wherein a concentration of the polysorbate in the composition is about 0.01% w/v to about 0.2% w/v.
 40. The composition of claim 35, wherein the protein of interest is selected from a group consisting of a monoclonal antibody, a polyclonal antibody, a bispecific antibody, an antibody fragment and an antibody-drug complex.
 41. The composition of claim 35 further comprising one or more pharmaceutically acceptable excipients.
 42. The composition of claim 35 further comprising a buffer selected from a group consisting of histidine buffer, citrate buffer, alginate buffer, and arginine buffer.
 43. The composition of claim 35 further comprising a tonicity modifier.
 44. The composition of claim 35, wherein concentration of the protein of interest is about 20 mg/mL to about 400 mg/mL.
 45. A composition having a protein of interest purified from mammalian cells, surfactant and a residual amount of liver carboxylesterase 1-like protein, wherein the residual amount of lysosomal acid lipase is less than about 5 ppm.
 46. The composition of claim 45, wherein the surfactant is polysorbate.
 47. The composition of claim 46, wherein the surfactant is polysorbate
 80. 48. The composition of claim 47, wherein the liver carboxylesterase 1-like protein causes degradation of the polysorbate
 80. 49. The composition of claim 46, wherein the composition is a parenteral formulation
 50. The composition of claim 46, wherein concentration of the polysorbate in the composition is about 0.01% w/v to about 0.2% w/v.
 51. The composition of claim 45, wherein the protein of interest is selected from a group consisting of a monoclonal antibody, a polyclonal antibody, a bispecific antibody, an antibody fragment and antibody-drug complex.
 52. The composition of claim 45 further comprising one or more pharmaceutically acceptable excipients.
 53. The composition of claim 45 further comprising a buffer selected from a group consisting of histidine buffer, citrate buffer, alginate buffer, and arginine buffer.
 54. The composition of claim 45 further comprising a tonicity modifier.
 55. The composition of claim 45, wherein concentration of the protein of interest is about 20 mg/mL to about 400 mg/mL. 